Methods and systems for generating nanoparticles

ABSTRACT

In one aspect, the present invention provides a process for forming polymeric nanoparticles, which comprises using a static mixer to create a mixed flowing stream of an anti-solvent, e.g., by introducing a liquid anti-solvent into a static mixer, and introducing a polymer solution into the mixed flowing anti-solvent stream such that controlled precipitation of polymeric nanoparticles occurs. The nanoparticles can then be separated from the anti-solvent stream.

RELATED APPLICATIONS

The present application claims priority as a continuation application toa patent application entitled “Methods and Systems for GeneratingNanoparticles” filed May 24, 2012, and having a patent application Ser.No. 13/479,646, which in turn claims priority as a continuationapplication to a patent application entitled “Methods and Systems forGenerating Nanoparticles” filed Feb. 8, 2011 and having a patentapplication Ser. No. 13/023,163 (now U.S. Pat. No. 8,207,290), which inturn claims priority to a provisional application entitled “Methods andSystems for Generating Nanoparticles” filed Mar. 26, 2010 and havingSer. No. 61/317,783, all of which are herein incorporated by referencein their entirety.

BACKGROUND

The present invention relates generally to methods, devices and systemsfor fabricating nanoparticles, and more particularly to such methods,devices and systems that can be employed to generate polymericnanoparticles.

A variety of methods are known for generating nanoparticles. In one suchmethod, commonly known as nanoprecipitation or flash precipitation, apolymer solution comprising a polymer dissolved in a process solvent isbrought into contact with another solvent (also known as anti-solvent)in which the process solvent is miscible but the polymer is not. As aresult, the process solvent diffuses rapidly into the anti-solvent whilethe polymer aggregates into a plurality of nanoparticles.

The conventional nanoprecipitation processes, however, suffer from anumber of shortcomings. For example, it is difficult to controlpredictably the average particle size and the size distribution of thegenerated nanoparticles. Further, many challenges exist in scaling upsuch processes to generate nanoparticles on a large scale.

Accordingly, there is a need for enhanced methods, devices and systemsfor generating nanoparticles.

SUMMARY

In one aspect, the present invention provides a process for formingpolymeric nanoparticles, which comprises introducing an anti-solventinto a static mixer to create a mixed flowing stream of the anti-solventand introducing a polymer-carrying liquid, e.g., a polymer solution, ora polymer dispersion or a mixed polymer solution/dispersion, into themixed flowing stream of the anti-solvent so as to form polymericnanoparticles. The polymeric nanoparticles can be formed vianon-reactive or reactive aggregation of at least one polymer, and insome cases one or more additives, of the polymer solution, or of thepolymer dispersion or of the mixed polymer solution/dispersion, as wellas in some embodiments a colloid stabilizer of the anti-solvent. Forexample, the polymeric nanoparticles can be formed via assembly/growthof at least one polymer, and in some cases one or more additives, of thepolymer solution, or of the polymer dispersion or of the mixed polymersolution/dispersion, as well as in some embodiments a colloid stabilizerof the anti-solvent. An example of reactive aggregation can includegenerating the polymeric nanoparticles via formation of covalentchemical bonds. An example of non-reactive aggregation can includegenerating the polymeric nanoparticles via assembly without formation ofcovalent chemical bonds.

For example, the nanoparticles can be formed by precipitation (e.g., acontrolled precipitation through selection of various parameters, suchas the flow rate of the anti-solvent and/or the flow rate and/or thepolymer concentration of the polymer solution (or of the polymerdispersion or of the mixed polymer solution/dispersion)). Thenanoparticles can then be separated from the anti-solvent stream.Although in the following description, various aspects and embodimentsof the invention are primarily described by reference to a polymersolution, the teachings of the invention can also be practiced with apolymer dispersion and/or a mixed polymer solution/dispersion.

The dimensions of the static mixer, e.g., its length and diameter, canvary over a wide range. By way of example, in some embodiments thestatic mixer can have a diameter greater than about 1 cm, or greaterthan about 2 cm, or greater than about 10 cm, or larger. For example,the static mixer can have a diameter in a range of about 1 cm to about100 cm, or in a range of about 20 cm to about 80 cm, or in a range ofabout 30 cm to about 70 cm, or in a range of about 40 cm to about 60 cm.In some embodiments, the static mixer can have between about 1 to about24 mixing elements. By way of example, the number of the mixing elementscan be in a range of about 12 to about 24. In some embodiments, thenumber of mixing elements is in a range of about 1 to about 4. In someembodiments, the static mixer is configured to cause substantiallyisotropic mixing of a fluid over at least about 50%, or at least about60%, or at least about 70%, or at least about 80%, or at least about90%, or over the entire volume of a portion of a conduit in which thestatic mixer is disposed.

A variety of flow rates, flow velocities and mixing conditions can beemployed. In some embodiments, the anti-solvent flowing stream isintroduced into the static mixer at a flow rate in a range of about 20ml/min to about 2000 ml/min, e.g., in a range of about 20 ml/min toabout 1500 ml/min, or in a range of about 30 ml/min to about 1000ml/min, or in a range of about 40 ml/min to about 500 ml/min, or in arange of about 20 ml/min to about 400 ml/min, or in a range of about 20ml/min to about 300 ml/min, or in a range of about 20 ml/min to about200 ml/min, or in a range of about 20 ml/min to about 100 ml/min. Insome embodiments, the anti-solvent flowing stream exhibits an averageaxial flow velocity in a range of about 1 cm/sec to about 100 cm/sec(e.g., in a range of about 1.5 cm/sec to about 60 cm/sec). By way ofexample, in some embodiments, the anti-solvent flowing stream canexhibit an average axial flow velocity in a range of about 1 cm/sec toabout 10 cm/sec, or in a range of about 10 cm/sec to about 20 cm/sec, orin a range of about 20 cm/sec to about 30 cm/sec, or in a range of about30 cm/sec to about 40 cm/sec, or in a range of about 40 cm/sec to about50 cm/sec, or in a range of about 50 cm/sec to about 60 cm/sec, or in arange of about 60 cm/sec to about 70 cm/sec, or in a range of about 70cm/sec to about 80 cm/sec, or in a range of about 80 cm/sec to about 90cm/sec, or in a range of about 90 cm/sec to about 100 cm/sec. In manyembodiments, the polymer solution is introduced into the mixed flowingstream of the anti-solvent as a liquid stream.

A wide range of ratios of the flow rate of the mixed flowing stream ofthe anti-solvent relative to that of the polymer solution stream can beemployed. For example, the ratio of the anti-solvent flow rate relativeto the polymer solution flow rate can be in a range of about 1:1 toabout 100:1, e.g., in a range of about 1:1 to about 10:1, or in a rangeof about 1:1 to about 20:1, or in a range of about 1:1 to about 30:1, orin a range of about 1:1 to about 40:1, or in a range of about 1:1 toabout 50:1, or in a range of about 1:1 to about 60:1, or in a range ofabout 1:1 to about 70:1, or in a range of about 1:1 to about 80:1, or ina range of about 1:1 to about 90:1. In some embodiments, the flow rateof the anti-solvent stream is about 10 times greater than the flow rateof the polymer solution stream. In some embodiments, the polymersolution is introduced into the mixed flowing stream of the anti-solventas a liquid stream at an axial flow velocity in a range of about 0.5cm/sec to about 40 cm/sec, for example, in a range of about 2 cm/sec toabout 20 cm/sec.

The nanoparticles can be formed via precipitation, typically over ashort time period, upon contact of the polymer solution with the mixedflowing stream of the anti-solvent. For example, the nanoparticles canbe generated via precipitation within a time period less than about 10milliseconds (e.g., a time period in a range of about 1 millisecond toabout 10 milliseconds, or in a range of about 2 milliseconds to about 10milliseconds), or within a time period less than about 5 milliseconds(e.g., a time period in a range of about 1 millisecond to about 5milliseconds, or a time period in a range of about 2 milliseconds toabout 5 milliseconds) upon exposure of the polymer solution to the mixedflowing stream of the anti-solvent. For example, in some embodiments, atleast about 50%, or at least about 60%, or at least about 70%, or atleast about 80%, or at least about 90%, or all of the nanoparticles areformed within a time period less than about 10 milliseconds (e.g., atime period in a range of about 1 millisecond to about 10 milliseconds,or a time period in a range of about 2 milliseconds to about 10milliseconds), or within a time period less than about 5 milliseconds(e.g., a time period in a range of about 1 millisecond to about 5milliseconds, or a time period in a range of about 2 milliseconds toabout 5 milliseconds) upon exposure of the polymer solution to the mixedflowing stream of the anti-solvent. In an embodiment, the time periodover which the nanoparticles are generated can be adjusted bycontrolling, e.g., the flow rate of the anti-solvent flowing stream, theconcentration of the polymer solution, the concentration of the colloidstabilizer, among others. For example, in an embodiment, as the flowrate of the anti-solvent flowing stream increases the time period overwhich the nanoparticles are generated decreases.

The polymer solution (and in some embodiments a polymer dispersion or amixed polymer solution/dispersion) can be introduced into the mixedflowing stream of the anti-solvent at a variety of locations. Forexample, the static mixer can extend from a proximal end to a distal endand the polymer solution can be introduced into the mixed flowing streamof the anti-solvent at an intermediate location between the proximal anddistal ends of the static mixer. Alternatively, the polymer solution canbe introduced into the mixed flowing stream of the anti-solvent inproximity to the proximal end of said static mixer. In otherembodiments, the polymer solution can be introduced into the mixedanti-solvent flowing stream in proximity to the distal end of the staticmixer.

In a related aspect, the nanoparticles generated by the above processexhibit a polydispersity index equal to or less than about 0.25. By wayof example, the nanoparticles can exhibit a polydispersity index in arange of about 0.05 to about 0.1.

In a related aspect, in the above process for fabricating nanoparticles,the flow rate of the mixed flowing stream of the anti-solvent can bechanged so as to adjust an average particle size of the polymericnanoparticles. By way of example, the flow rate of the anti-solventstream can be selected such that the polymeric nanoparticles exhibit anaverage particle size equal to or less than about 200 nm whileexhibiting in some cases a particle size distribution less than about100 nm. Further, in some embodiments, the flow rate of the anti-solventstream can be selected such that the polymeric nanoparticles willexhibit an average particle size equal to or less than about 100 nm,e.g., in a range of about 40 nm to about 100 nm. By way of example, insome embodiments, the flow rate of the mixed flowing stream of theanti-solvent can be varied between about 100 ml/min to about 1800 ml/minto adjust the average particle size of the polymeric nanoparticles in arange of about 100 nm to about 230 nm.

In a related aspect, the flow rate of the mixed flowing stream of theanti-solvent can be selected to be in a range in which an averageparticle size of the polymeric nanoparticles is substantiallyindependent of the anti-solvent flow rate. Alternatively, the flow rateof mixed flowing stream of the anti-solvent can be selected to be in arange in which an average particle size of the polymeric nanoparticlesis strongly dependent on the anti-solvent flow rate. For example, in anembodiment, when the flow rate of the mixed flowing stream of theanti-solvent is less than about 200 ml/min, e.g., in a range of about 20ml/min to about 200 ml/min, or in a range about 20 ml/min to about 100ml/min, the average particle size of the polymeric nanoparticles isstrongly dependent on the anti-solvent flow rate. For example, in anembodiment, when the flow rate of the mixed flowing stream of theanti-solvent is greater than about 200 ml/min, e.g., greater than about300 ml/min (e.g., in a range of about 300 ml/min to about 1000 ml/min,or in a range of about 500 ml/min to about 2000 ml/min), the averageparticle size of the polymeric nanoparticles is substantiallyindependent of the anti-solvent flow rate.

In a related aspect, the average axial flow velocity of the mixedflowing stream of the anti-solvent or that of the polymer solution canbe selected to be in a range in which an average particle size of thenanoparticles is substantially independent of such axial flow velocity.Alternatively, the average axial flow velocity of the mixed flowingstream of the anti-solvent or that of the polymer solution can beselected to be in a range in which an average particle size of thenanoparticles is strongly dependent on such flow velocity.

In another aspect, a ratio of a flow rate of the anti-solvent streamrelative to a flow rate of the polymer solution can be changed so as toadjust an average particle size of the polymeric nanoparticles.

In some embodiments, the method for forming polymeric nanoparticles caninclude the additional steps of selecting one or more parameters, e.g.,anti-solvent and/or polymer solution flow rate, polymer concentration inthe polymer solution, the average axial flow velocity of the mixedflowing stream of the anti-solvent and/or that of the polymer solution,or other parameters discussed herein, and carrying out the method undersuch selected conditions. Optionally, the method can include evaluatinga sample of the nanoparticles produced to determine if the nanoparticlesmeet one or more predefined criteria, e.g., average particle size,polydispersity, drug loading, etc. In some embodiments, if the sample ofthe nanoparticles fails to meet the one or more predefined criteria, oneor more of the parameters, such as those listed above, can be adjustedand the method carried out under the adjusted conditions. Again, asample of the nanoparticles produced can be evaluated to determine ifthe nanoparticles meet the one or more predefined criteria. This processcan be repeated, if needed, until a sample of the nanoparticles thatmeets the one or more predefined criteria is achieved.

In some embodiments, at least one attribute of a sample of nanoparticlesproduced (e.g., an average particle size, polydispersity, drug loading,etc), or that of its preparation, can be compared with a reference valuefor that attribute. The reference value can be, e.g., a releaseparameter or a manufacturing specification, e.g., one set by aregulatory agency, e.g., the FDA or EMEA, a compendial authority, or amanufacturer. In an embodiment, the reference value is a value exhibitedby a preparation previously made by the method. In an embodiment, e.g.,responsive to whether the attribute meets a reference value for thatattribute a further decision or step is taken, e.g., the sample isclassified, selected, rejected, accepted, or discarded, released orwithheld, processed into a drug product, shipped, moved to a differentlocation, formulated, labeled, packaged, released into commerce,exported, imported, or sold or offered for sale, depending on whetherthe preselected criterion is met. For example, based on the result ofthe evaluation, the batch from which a sample is taken can be processed,e.g., as just described. For example, if the criterion is met, thepreparation is sold, shipped, or offered for sale or otherwise releasedinto commerce.

The polymer solution can comprise a polymer dissolved in a processsolvent, wherein the process solvent is miscible, or at least partiallymiscible, with the anti-solvent. In some embodiments, the concentrationof the polymer in the polymer solution can be changed so as to adjust anaverage particle size of the polymeric nanoparticles. A variety ofpolymers can be employed. By way of example, the polymer can be any ofpoly(lactide-co-glycolide), poly(lactide), poly(epsilon-caprolactone),poly(isobutylcyanoacrylate), poly(isohexylcyanoacrylate),poly(n-butylcyanoacrylate), poly(acrylate), poly(methacrylate),poly(lactide)-poly(ethylene glycol),poly(lactide-co-glycolide)-poly(ethylene glycol),poly(epsilon-caprolactone)-poly(ethylene glycol), andpoly(hexadecylcyanoacrylate-co-poly(ethylene glycol) cyanoacrylate).

In some embodiments, the polymer solution can include at least oneadditive. The additive can be any of a therapeutic agent or an imagingagent. In some embodiments, such a therapeutic or imaging agent can becoupled to, associated with, or incorporated in the polymer. Forexample, in some embodiments, such a therapeutic or imaging agent can beconjugated to, or embedded in the polymer. In some embodiments, multipledifferent agents can be coupled to, associated with, or incorporated inthe polymer. In some embodiments, the imaging agent can be coupled tothe therapeutic agent

By way of example, the therapeutic agent can be, without limitation, anyof an anti-neoplastic agent, an anti-inflammatory agent, acardiovascular active agent, or an anti-metabolite.

In some embodiments, the therapeutic agent can be any of a taxane, anepothilone, a boronic acid proteasome inhibitor, and an antibiotic.

In some embodiments, the imaging agent can be, without limitation, anyof a radioactive or non-radioactive agent, or a fluorescent agent. Someexamples of suitable imaging agents include, without limitation,Technetium Bicisate, Ioxaglate, Fluorodeoxyglucose, label-free Ramanimaging agents, encapsulate MRI contrast agent Gd-DTPA, and rhodamine 6Gas a fluorescent agent. In some embodiments, the imaging agent can beradiolabeled docetaxel (e.g., 3H-radiolabeled docetaxel or14C-radiolabeled docetaxel), or radiolabeled paclitaxel.

The process solvent can include, without limitation, any of acetone,ether, alcohol, tetrahydrofuran, 2-pyrrolidone, N-Methyl-2-pyrrolidone(NMP), dimethylformamide (DMF), dimethylacetamide (DMA), methyl acetate,ethyl formate, methyl ethyl ketone (MEK), methyl isobutyl ketone (MIBK),methyl propyl ketone, isopropyl ketone, isopropyl acetate, acetonitrile(MeCN) and dimethyl sulfoxide (DMSO).

In some embodiments, the anti-solvent can include an aqueous solution.By way of example, the aqueous solution can include any of an alcohol oran ether, and water. In some embodiments, the anti-solvent can includean organic solvent or a mixture of two or more organic solvents. Forexample, the anti-solvent can include, without limitation, any ofmethanol, ethanol, n-propanol, isopropanol, n-butanol, and ethyl ether.

In some embodiments, the anti-solvent can include a colloid stabilizer.By way of example, the colloid stabilizer can include, withoutlimitation, any of poly(vinyl alcohol), Dextran and pluronic F68, poly(vinyl pyrrolidone), solutol, Tween 80, poloxamer, carbopol,poly-ethylene glycol, sodium dodecyl sulfate, poly (ε-caprolactone),peptides, and carbohydrates.

In some embodiments, the polymer solution is delivered as a liquidstream that intersects the anti-solvent stream at a non-zero angle. Theangle can be an acute angle, for example, one in a range of about 10degrees to about 90 degrees (e.g., in a range of about 50 degrees toabout 90 degrees). In some embodiments, the angle can be in a range ofabout 10 degrees to about 170 degrees. In some other embodiments, thepolymer solution is injected into the flowing stream of theanti-solvent.

In another aspect, the step of separating the nanoparticles includescollecting the nanoparticles downstream from the static mixer as asuspension in a mixture of the anti-solvent and a process solvent of thepolymer solution. At least a portion of the process solvent can beremoved from the suspension in order to concentrate the suspension. Forexample, the suspension can be diafiltered to remove at least a portionof the process solvent.

In some embodiments, a lyoprotectant can be added to the preparation,e.g., the suspension. It can be added prior to or after the step ofconcentrating the suspension, to protect the nanoparticles in asubsequent lyophilization step. By way of example, the lyoprotectant canbe, without limitation, a derivatized cyclic oligosaccharide, e.g., aderivatized cyclodextrin, e.g., 2 hydroxy propyl-β cyclodextrin, e.g.,partially etherified cyclodextrins (e.g., partially etherified βcyclodextrins) disclosed in U.S. Pat. No. 6,407,079, the contents ofwhich are incorporated herein by this reference.

In another aspect, a process for forming polymeric nanoparticles isdisclosed, which includes introducing an anti-solvent into a staticmixer so as to generate a mixed flowing stream of the anti-solvent, andintroducing a polymer solution (or a polymer dispersion or a mixedpolymer solution/dispersion) into the mixed flowing stream of theanti-solvent to generate polymeric nanoparticles (e.g., viaprecipitation) such that the polymeric nanoparticles exhibit apolydispersity index equal to or less than about 0.25. For example, thepolymeric nanoparticles can exhibit a polydispersity index in a range ofabout 0.05 to about 0.1.

In some embodiments, the polymeric nanoparticles can exhibit an averageparticle size equal to or less than about 500 nm. For example, thepolymeric nanoparticles can exhibit an average particle size in a rangeof about 5 nm to about 500 nm, or in a range of about 10 nm to about 500nm, or in a range of about 20 nm to about 500 nm, or in a range of about30 nm to about 500 nm, or in a range of about 40 nm to about 500 nm, orin a range of about 50 nm to about 500 nm.

In some embodiments, the polymeric nanoparticles can exhibit an averageparticle size equal to or less than about 400 nm. For example, thepolymeric nanoparticles can exhibit an average particle size in a rangeof about 5 nm to about 400 nm, or in a range of about 10 nm to about 400nm, or in a range of about 20 nm to about 400 nm, or in a range of about30 nm to about 400 nm, or in a range of about 40 nm to about 400 nm, ina range of about 50 nm to about 400 nm.

In some embodiments, the polymeric nanoparticles can exhibit an averageparticle size equal to or less than about 300 nm. For example, thepolymeric nanoparticles can exhibit an average particle size in range ofabout 5 nm to about 300 nm, or in a range of about 10 nm to about 300nm, or in a range of about 20 nm to about 300 nm, or in a range of about30 nm to about 300 nm, or in a range of about 40 nm to about 300 nm, orin a range of about 50 nm to about 300 nm.

In some embodiments, the polymeric nanoparticles can exhibit an averageparticle size equal to or less than about 200 nm. For example, thepolymeric nanoparticles can exhibit an average particle size in a rangeof about 5 nm to about 200 nm, or in a range of about 10 nm to about 200nm, or in a range of 20 nm to about 200 nm, or in a range of about 30 nmto about 200 nm, or in a range of about 40 nm to about 200 nm, or in arange of about 50 nm to about 200 nm.

In some embodiments, the polymeric nanoparticles can exhibit an averageparticle size equal to or less than about 100 nm. For example, thepolymeric nanoparticles can exhibit an average particle size in a rangeof about of 5 nm to about 100 nm, or in a range of about 10 nm to about100 nm, or in a range of about 20 nm to about 100 nm, or in a range ofabout 30 nm to about 100 nm, or in a range of about 40 nm to about 100nm, or in a range of about 50 nm to about 100 nm.

In some embodiments, the anti-solvent flow comprises a stream exhibitinga flow rate in a range of about 20 ml/min to about 2000 ml/min. In someembodiments, the mixed flowing stream of anti-solvent exhibits anaverage axial velocity in a range of about 1 cm/sec to about 100 cm/sec,e.g., in a range of about 1.5 cm/sec to about 60 cm/sec.

In the above process for forming polymeric nanoparticles, the polymersolution can be introduced into the mixed flowing stream of theanti-solvent at a variety of locations relative to the static mixer. Forexample, the polymer solution can be introduced into the mixed flowingstream of the anti-solvent at an intermediate location between aproximal end and a distal end of the static mixer. Alternatively, thepolymer solution can be introduced into the mixed flowing stream of theanti-solvent in proximity to the proximal end, or the distal end, of thestatic mixer.

In the above process, the polymer solution can be introduced as a liquidstream into the mixed flowing stream of the anti-solvent at a variety offlow rates. For example, a flow rate of the anti-solvent stream relativeto a flow rate of said polymer solution stream can be in a range ofabout 1:1 to about 100:1, e.g., in a range of about 1:1 to about 10:1,or in a range of about 1:1 to about 20:1, or in a range of about 1:1 toabout 30:1, or in a range of about 1:1 to about 40:1, or in a range ofabout 1:1 to about 50:1, or in a range of about 1:1 to about 60:1, or ina range of about 1:1 to about 70:1, or in a range of about 1:1 to about80:1, or in a range of about 1:1 to about 90:1. Further, in someembodiments, the polymer solution stream is introduced into the mixedflowing stream of the anti-solvent at a non-zero angle, e.g., an acuteangle, relative to a flow direction of the anti-solvent stream. In someembodiments, the polymer solution is injected into the mixedanti-solvent stream.

The polymer solution can include a polymer dissolved in a processsolvent, where the process solvent is miscible, or is at least partiallymiscible, with the anti-solvent. In some embodiments, the polymersolution can include at least one additive, such as a therapeutic agentor an imaging agent. A variety of therapeutic agents and imaging agentscan be employed, such as those listed above. In some embodiments, one ormore of such agents are coupled to, associated with, or incorporated inthe polymer. In some embodiments, multiple different agents can becoupled to, associated with, or incorporated in the polymer. In someembodiments, one or more of such agents are conjugated to, or embeddedin the polymer.

A variety of polymers, process solvents and anti-solvents can beemployed in the above process. Some examples of such polymers, processsolvents and anti-solvents are provided above. In some embodiments, theanti-solvent can include a colloid stabilizer, such as those listedabove.

In another aspect, the invention provides a process for controllingparticle size of nanoparticles formed, e.g., by precipitation, whichcomprises introducing an anti-solvent liquid flow into a static mixer togenerate a mixed flowing stream of the anti-solvent, and introducing apolymer solution into the mixed flowing stream of the anti-solvent so asto generate a plurality of polymeric nanoparticles, e.g., byprecipitation. The flow rate of the anti-solvent stream through saidstatic mixer is controlled so as to adjust an average particle size ofthe nanoparticles.

The step of controlling the flow rate of the anti-solvent stream caninclude changing the flow rate so as to vary the average particle sizein a range of about 50 nm to about 200 nm.

In the above process for controlling particle size of nanoparticles, thepolymer solution can comprise a polymer dissolved in a process solventthat is miscible, or at least partially miscible, in the anti-solvent.In some embodiments, the polymer solution can include an additive, suchas a therapeutic or an imaging agent. In some embodiments, one or moreof such agents are embedded in the polymer. In some embodiments, one ormore of such agents are conjugated to the polymer. Some examples ofsuitable therapeutic and imaging agents are provided above.

A variety of polymers, process solvents and anti-solvents can beemployed in the above process. Some examples of such polymers, processsolvents and anti-solvents are provided above.

In some embodiments, the anti-solvent can include a colloid stabilizer.Some examples of suitable colloid stabilizers are provided above.

In another aspect, a system for generating polymeric nanoparticles isdisclosed, which comprises a conduit having a first input port forreceiving an anti-solvent, and at least one static mixer disposed in theconduit to generate a mixed flowing stream of the anti-solvent, wherethe static mixer extends from a proximal end to a distal end. Theconduit has a second input port disposed relative to the static mixer soas to allow introducing a polymer solution into the mixed flowing streamof the anti-solvent to generate polymeric nanoparticles, e.g., viaprecipitation. The system can further include a device, e.g., a variablepump, adapted to cause a flow of the anti-solvent from a reservoir tothe conduit and to control a flow rate of the anti-solvent through thestatic mixer for adjusting an average particle size of thenanoparticles.

In some embodiments, the conduit in which the static mixer is disposedhas an internal diameter of at least about 1 mm, or at least about 10mm, or at least about 100 mm, or at least 500 mm.

In some embodiments, the device for causing the anti-solvent flow isadapted to control a flow rate of said anti-solvent through the conduitwithin a range of about 20 ml/min to about 2000 ml/min.

In some embodiments, the second input port is located at an intermediatelocation between the proximal and distal ends of the static mixer. Insome other embodiments, the second input port is located in proximity tothe proximal end, or the distal end, of the static mixer. In someembodiments, the second input port is configured so as to allowintroduction of the polymer solution into the conduit at a non-zeroangle, e.g., at an acute angle (e.g., wherein the angle between thedirection of flow through the conduit and the direction of flow enteringthe conduit through the second input port is in a range of about 50degrees to about 90 degrees), relative to a flow direction of theanti-solvent stream.

In some embodiments, the system includes at least one injector coupledto the second input port for injecting the polymer solution into themixed flowing stream of the anti-solvent.

In some embodiments, the system can further include a reservoir forcontaining a quantity of the polymer solution. A device adapted to causea flow of the polymer solution, e.g., a pump, can cause the polymersolution to flow from the reservoir through the second input port intothe conduit. The device can be capable of adjusting the flow rate of thepolymer solution through the second port. For example, the device can beadapted to control the flow rate of the polymer solution through thesecond input port in a range of about 4 ml/min to about 200 ml/min, forexample, in a range of about 5 ml/min to about 100 ml/min.

In the above system, the conduit can comprise an output port throughwhich the polymeric nanoparticles exit the conduit as a suspension in amixture of the anti-solvent and a process solvent of the polymersolution. A collection vessel coupled to the output port of the conduitcan collect the suspension containing the nanoparticles. The collectionvessel can contain a liquid. In many embodiments, a stirrer is disposedin the collection vessel for mixing the liquid.

In another aspect, a device for generating nanoparticles is disclosed,which comprises a conduit having a first input port for receiving astream of an anti-solvent and an output port. A static mixer is disposedin the conduit to cause mixing of the anti-solvent stream to generate amixed flowing stream of the anti-solvent, where the static mixer extendsfrom a proximal end to a distal end. The conduit has a second input portpositioned relative to the static mixer so as to allow delivery of apolymer solution into said mixed flowing stream of the anti-solvent forgenerating polymeric nanoparticles, e.g., by precipitation.

In some embodiments, the second input port is positioned at anintermediate location relative to the proximal and distal ends of thestatic mixer. In some alternative embodiments, the second input port ispositioned in proximity to the proximal end, or the distal end, of thestatic mixer. In some embodiments, the second input port is positioneddownstream from the static mixer and sufficiently close to the mixer toallow the delivery of the polymer solution into the mixed flowing streamof the anti-solvent.

In some embodiments, the second input port is configured so as tointroduce the polymer solution into the anti-solvent stream at anon-zero angle, e.g., at an acute angle, relative to a flow direction ofthe mixed flowing stream of the anti-solvent. In some embodiments, theangle can be in a range of about 50 degrees to about 90 degrees.

In some embodiments, the device can further include a collection tank influid communication with the output port for receiving a suspensioncontaining the polymeric nanoparticles. In some embodiments, the tankcan store a quantity of an aqueous solution.

In a related aspect, the above device contains the anti-solvent and/orthe polymer solution discussed above.

In another aspect, a device for generating nanoparticles is disclosed,which comprises a conduit having a first input port for receiving astream of a liquid anti-solvent and an output port. A static mixer isdisposed in the conduit to cause mixing of the anti-solvent stream togenerate a mixed flowing stream of the anti-solvent, where the staticmixer extends from a proximal end to a distal end. The device furtherincludes an injector coupled to the conduit for injecting a polymersolution into the mixed flowing stream of the anti-solvent.

In some embodiments, the injector is positioned so as to inject thepolymer solution at an intermediate location between the proximal anddistal ends of the static mixer. Alternatively, the injector can bepositioned to inject the polymer solution in proximity to the proximal,or the distal, end of the static mixer. In some embodiments, theinjector is configured to inject the polymer solution into the mixedflowing stream of the anti-solvent along a direction substantiallyparallel to the flow direction of the anti-solvent.

In a related aspect, the device contains the anti-solvent and/or thepolymer solution discussed above.

In another aspect, a process for forming polymeric nanoparticles isdisclosed, which comprises using a static mixer to create a mixedflowing stream of an anti-solvent, and introducing a polymer solutioninto the mixed flowing stream of the anti-solvent such that controlledprecipitation of polymeric nanoparticles occurs. In some embodiments, aflow rate of the mixed flowing stream of the anti-solvent and/or that ofthe polymer solution can be controlled so as to adjust an averageparticle size of the nanoparticles.

In another aspect, a process for monitoring nanoparticles formed byintroducing a polymer solution into a mixed flowing stream of ananti-solvent is disclosed, which includes selecting one or moreparameters, such as, the flow rate of the anti-solvent stream, thepolymer solution flow rate, the concentration of polymer in the polymersolution, and the concentration of a colloid stabilizer in theanti-solvent. The polymer solution is introduced, under such conditions,into a mixed flowing stream of the anti-solvent, which is created byintroducing the anti-solvent into a static mixer, so as to formpolymeric nanoparticles, e.g., by precipitation. The nanoparticlesproduced are then examined to determine if one or more of theirattributes (e.g., their average particle size or polydispersity index)meet one or more predefined criteria. If they do not, one or more of theabove parameters are adjusted, and polymer solution is introduced, underthe adjusted conditions, into the mixed flowing stream of theanti-solvent to generate a new population of nanoparticles. The newpopulation of the nanoparticles can be examined to determine if one ormore of their attributes meet the predefined criteria. The above stepsare repeated until a population of nanoparticles whose one or moreattributes meet the predefined criteria is achieved.

In another aspect, a plurality of polymeric nanoparticles are generatedby using the above processes.

In another aspect, a population of polymeric nanoparticles having anaverage particle size in a selected range, e.g., one of the rangesdescribed above, is generated by using the above processes.

In another aspect, a population of polymeric nanoparticles having apolydispersity index less than about 0.25, e.g., in a range of about0.05 to about 0.1, is generated by using the above processes.

In a related aspect, a population of polymeric nanoparticles thatincludes at least about 10 grams, or at least about 20 grams, or atleast about 30 grams, or at least about 40 grams, or at least about 50grams, or at least about 100 grams, or at least about 200 grams, or atleast about 300 grams, or at least about 400 grams, or at least about500 grams, or at least about 1000 grams of the nanoparticles isgenerated by using the above processes.

In a related aspect, a population of polymeric nanoparticles havingpoly(lactic-co-glycolic acid) (PLGA) as at least one polymeric componentis generated by using the above processes. In some embodiments, the PLGApolymer is attached to a therapeutic agent. For example, the therapeuticagent can be an anti-neoplastic agent. In some embodiments, theanti-neoplastic agent is a taxane (e.g., paclitaxel, docetaxel,larotaxel, or cabazitaxel).

In another aspect, a pharmaceutically acceptable preparation ofpolymeric nanoparticles is generated by using the above processes. In anembodiment, the pharmaceutically acceptable preparation includes, e.g.,a pharmaceutically acceptable excipient, e.g., a lyoprotectant. In anembodiment, the pharmaceutically acceptable preparation is a liquid or alyophilized powder.

In an embodiment, a process described herein further includes dividing afirst pharmaceutically acceptable preparation made by a processdescribed herein into smaller aliquots and optionally packaging aplurality of aliquots into gas and/or liquid tight containers.

In an embodiment, a process described herein further includes testingthe product (e.g., the preparation of the nanoparticles) to determine ifit meets a preselected reference value, e.g., a value for concentration,average particle size, purity, polydispersity index, or other particleproperties described herein.

Further understanding of the invention can be obtained by reference tothe following detailed description in conjunction with the associateddrawings, which are described briefly below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow chart depicting various steps in an exemplaryembodiment of a method according to the invention for generatingpolymeric nanoparticles,

FIG. 2 is a flow chart depicting various steps according to an exemplaryembodiment of the invention for performing a precipitation process forgenerating polymeric nanoparticles,

FIG. 3 is a flow chart depicting various steps according to an exemplaryembodiment of the invention for controlling particle size ofnanoparticles formed by nanoprecipitation,

FIG. 4A schematically depicts a device according to an exemplaryembodiment of the invention for forming nanoparticles,

FIG. 4B schematically depicts an alternative implementation of thedevice of FIG. 4A,

FIG. 5A schematically depicts a device according to another embodimentfor generating nanoparticles in accordance with the teachings of theinvention,

FIG. 5B schematically depicts a device according to another embodimentfor generating nanoparticles in accordance with the teachings of theinvention,

FIG. 5C schematically depicts a device according to another embodimentfor generating nanoparticles in accordance with the teachings of theinvention,

FIG. 5D schematically depicts a device according to another embodimentfor generating nanoparticles in accordance with the teachings of theinvention,

FIG. 5E schematically depicts a device according to another embodimentfor generating nanoparticles in accordance with the teachings of theinvention,

FIG. 6A schematically depicts a device according to another embodimentfor generating nanoparticles in accordance with the teachings of theinvention in which a plurality of static mixer units are employed,

FIG. 6B schematically depicts a device according to an alternativeimplementation of the device of FIG. 6A,

FIG. 6C schematically depicts a device according to another embodimentof the invention,

FIG. 7 schematically depicts a system according to an exemplaryembodiment of the invention for generating nanoparticles,

FIG. 8A schematically depicts a device according to an embodiment of theinvention for generating nanoparticles, which employs an injector forinjecting a polymer solution into a mixed flowing stream of ananti-solvent,

FIG. 8B schematically depicts a device according to another embodimentof the invention for generating nanoparticles, which employs aninjection system disposed across a conduit through which a mixed streamof an anti-solvent flows to inject a polymer solution into theanti-solvent flow,

FIG. 9 shows a helical static mixer employed in some prototype devicesbased on the teachings of the invention for generating nanoparticles,

FIGS. 10A and 10B provide two views of a structured-packing mixermarketed by Sulzer Chemtech USA, Inc. of Oklahoma, U.S.A. under thetrade designation Sulzer SMX, which was employed in some prototypedevices based on the teachings of the invention for generatingnanoparticles,

FIG. 11A shows a prototype device according to an embodiment of theinvention, which was employed to generate polymeric nanoparticles,

FIG. 11B shows a prototype device according to an embodiment of theinvention, which was employed to generate polymeric nanoparticles,

FIG. 12 presents data corresponding to Z_(ace) a function of total flowrate for nanoparticles generated in a prototype device according to theteachings of the invention,

FIG. 13 presents data corresponding to Z_(ave) as a function ofanti-solvent flow rate for nanoparticles generated in two prototypedevices according to the teachings of the invention, and

FIG. 14 shows particle size distribution exhibited by polymericnanoparticles generated by employing a prototype device according to theteachings of the invention, and

FIG. 15 shows three graphs corresponding to three measurements of theparticle size distribution exhibited by a plurality of polymericnanoparticles generated by employing a prototype device according to theteachings of the invention as discussed below in Example 5.

DETAILED DESCRIPTION

The present invention relates generally to methods, devices and systemsfor generating nanoparticles, e.g., polymeric nanoparticles. In someembodiments, the nanoparticles are formed by introducing a polymersolution, which comprises one or more polymer(s) dissolved in a processsolvent, into a mixed flowing stream of an anti-solvent, which ismiscible, or at least partially miscible, with the process solvent butin which the polymer(s) cannot be dissolved in any appreciable amount,to cause precipitation of the polymer(s) into a plurality ofnanoparticles. As discussed in more detail below, it has been discoveredthat utilizing a static mixer to generate a mixed flowing stream of theanti-solvent, and introducing the polymer solution into such a mixedflowing stream to cause precipitation can provide significantadvantages. For example, it allows forming the nanoparticles with a lowpolydispersity index over a wide range of anti-solvent (and polymersolution) flow rates. It has also been discovered that the anti-solventflow rate and/or the polymer solution flow rate can be changed to adjustthe average particle size of the fabricated nanoparticles. The lowpolydispersity index exhibited by the nanoparticles can be beneficial ina variety of applications such as pharmaceutical applications. Moreover,the precipitation process can be scaled up to form nanoparticles on alarge scale.

The following definitions are provided for a variety of terms andphrases utilized herein:

Static Mixer:

The term “static mixer” or “motionless mixer” as used herein refers to adevice that includes one or more substantially stationary mixingelements, e.g., baffles such as blades, plates, vanes, that are adaptedfor placement in the path of a flowing fluid, e.g., a fluid flowingthrough a conduit, to produce a pattern of flow divisions and splits toaccomplish mixing, e.g., radial mixing via radial circulation orexchange, in the flowing fluid. Although the stationary mixing elementsare typically immovable within the conduit, some limited movement of thestationary elements relative to the conduit can occur so long as suchlimited movement does not contribute substantially to the mixing of theflowing fluid. In a static mixer having multiple stationary elements,these elements are typically arranged in series and in a staggeredorientation relative to one another.

Mixed Flowing Stream:

The term “mixed flowing stream” as used herein refers to a flowingstream of a fluid, e.g., a liquid, that exhibits active motion normal toits direction of flow.

Polymer Solution:

The term “polymer solution” as used herein refers to a solutioncomprising one or more polymers dissolved in a liquid solvent, which isherein also referred to as process solvent. The polymer(s) are typicallysufficiently soluble in the solvent such that a concentration of atleast about 0.1 percent by weight, and preferably at least about 0.2percent by weight, of the polymer(s) can be dissolved in the solvent atroom temperature. In some cases, the concentration of the polymer(s)that can be dissolved in the solvent at room temperature can beoptionally less than about 10 percent by weight, e.g., less than about 5percent by weight. The polymer solution can also include a variety ofadditives, such as therapeutic and/or imaging agents or othersupplemental additives useful for the production and/or subsequent useof the nanoparticles.

Anti-Solvent:

The term “anti-solvent” as used herein refers to a liquid, or a mixtureof liquids, which is incapable of dissolving any appreciableconcentration (e.g., a concentration equal to or greater than about 0.1%at room temperature) of the polymer(s) of the polymer solution, but ismiscible, or at least partially miscible, with the process solvent. Insome embodiments, the anti-solvent and the process solvent can be mixedin all proportions to form a homogeneous solution. When combined withthe polymer solution, the anti-solvent causes at least a portion of thepolymer to precipitate.

Average Axial Flow Velocity:

The phrase “average axial flow velocity” as used herein refers to avelocity of a fluid, e.g., liquid, along the direction of flow averagedover a cross-sectional area of the flow, e.g., averaged over across-sectional area of a conduit through which the fluid flows. Theaverage axial flow velocity (V_(ave)) can be obtained by the followingrelation:

$\begin{matrix}{V_{ave} = \frac{Q}{A}} & {{Eq}.(1)}\end{matrix}$wherein,

Q represent the volumetric rate of fluid flow along the direction offlow (e.g., in units of ml/sec), and

A represents a cross-sectional area of the flow, e.g., a cross-sectionalarea of a conduit through which the fluid flows.

Nanoparticle:

The term “nanoparticle” is used herein to refer to a material structurewhose size in any dimension (e.g., x, y, and z Cartesian dimensions) isless than about 1 micrometer (micron), e.g., less than about 500 nm orless than about 200 nm or less than about 100 nm, and greater than about5 nm. A nanoparticle can have a variety of geometrical shapes, e.g.,spherical, ellipsoidal, etc. The term “nanoparticles” is used as theplural of the term “nanoparticle.”

Average Particle Size:

The term “average particle size” is a length dimension which isdesignated herein as Z average or Z_(ave), and as used herein refers tothe intensity weighted mean hydrodynamic size of an ensemble collectionof particles measured by dynamic light scattering (DLS). The Z averageis derived from a Cumulants analysis of a measured autocorrelationcurve, wherein a single particle size is assumed and a singleexponential fit is applied to the autocorrelation function. Theautocorrelation function (G(τ)) is defined as follows:

$\begin{matrix}{{{G(\tau)} = {\left\langle {{I(t)} \cdot {I\left( {t + \tau} \right)}} \right\rangle = {A\left\lbrack {1 + {B\;{\exp\left( {{- 2}\;\Gamma\;\tau} \right)}}} \right\rbrack}}}{{wherein},}} & {{Eq}.\mspace{14mu}(3)} \\{\Gamma = {Dq}^{2}} & {{Eq}.\mspace{14mu}(4)} \\{q = {\frac{4\;\pi\;\overset{\sim}{n}}{\lambda_{0}}{\sin\left( \frac{\theta}{2} \right)}}} & {{Eq}.\mspace{14mu}(5)} \\{{D = \frac{kT}{6\;\pi\;\mu\; R_{H}}},} & {{Eq}.\mspace{14mu}(6)}\end{matrix}$wherein,

I represents the light scattering intensity,

t represents an initial time,

-   -   τ represents the delay time,

A represents an amplitude (or intercept) of the autocorrelationfunction,

B represents the baseline,

D represents the diffusion coefficient,

q represents the scattering vector,

k represents the Boltzmann constant,

λ₀ represents the vacuum wavelength of a laser source employed for thelight scattering measurements,

ñ represents the index of refraction of the medium,

-   -   θ represents the scattering angle,

T represents the absolute temperature (Kelvin),

μ represents the viscosity of the medium, and

R_(H) represents the hydrodynamic radius.

In the Cumulants analysis, the exponential fitting expression of Eq. (3)is expanded as indicated below as expression y(τ) in Eq. (7) to accountfor polydispersity, which is defined in more detail below, or peakbroadening,

$\begin{matrix}{{y(\tau)} = {{\frac{1}{2}{\ln\left\lbrack {{G(\tau)} - A} \right\rbrack}} = {{{\frac{1}{2}{\ln\left\lbrack {{AB}\;{\exp\left( {{{- 2}\Gamma\;\tau} + {\mu_{2}\tau^{2}}} \right)}} \right\rbrack}} \cong {{\frac{1}{2}{\ln\lbrack{AB}\rbrack}} - {\left\langle \Gamma \right\rangle\tau} + {\frac{\mu_{2}}{2}\tau^{2}}}} = {a_{0} - {a_{1}\tau} + {a_{2}\tau^{2}}}}}} & {{Eq}.\mspace{14mu}(7)}\end{matrix}$

wherein μ₂ is a fitting parameter and the other parameters are definedabove.

The dynamic light scattering data can be fit to the above expression(Eq. (7)) to obtain values of the parameters a₀, a₁, and a₂. The firstCumulant moment (a₁) can be utilized to obtain Z_(ave) as follows:

$\begin{matrix}{Z_{ave} = {\frac{1}{a_{1}}{\frac{kT}{3\;\pi\;\mu}\left\lbrack {\frac{4\;\pi\;\overset{\sim}{n}}{\lambda_{0}}{\sin\left( \frac{\theta}{2} \right)}} \right\rbrack}^{2}}} & {{Eq}.\mspace{14mu}(8)}\end{matrix}$wherein the parameters are defined above.

The first Cumulant moment (a₁) and the second Cumulant moment (a₂) canbe used to calculate another parameter known as polydispersity index(PdI), which is discussed in more detail below, as follows:

$\begin{matrix}{{PdI} = \frac{2a_{2}}{a_{1}^{2}}} & {{Eq}.\mspace{14mu}(9)}\end{matrix}$Polydispersity Index:

The term “polydispersity index” is used herein as a measure of the sizedistribution of an ensemble of particles, e.g., nanoparticles. Thepolydispersity index is calculated as indicated in the above Eq. (9)based on dynamic light scattering measurements.

Particle Size Distribution:

If it is assumed that an ensemble of particles exhibit a Gaussian sizedistribution, then the particle size distribution of such an ensemble isa length dimension that can be defined as the square root of thestandard deviation of the Gaussian distribution (σ²) as follows:σ² =PdI·Z _(ave) ²  Eq. (10)Particle Size Distribution=√{square root over (σ²)}  Eq. (11)wherein Z_(ave) is defined by Eq. (8) above.Colloid Stabilizer:

The term colloid stabilizer as used herein refers to an additive addedto the anti-solvent and/or the polymer solution to prevent or retard anunwanted alteration of the physical state of the particles, e.g., acolloid stabilizer can inhibit aggregation of the nanoparticles. Forexample, a colloid stabilizer can inhibit aggregation of thenanoparticles during and/or after their formation.

Lyoprotectant:

The term “lyoprotectant,” as used herein refers to a substance presentin a lyophilized preparation. Typically it is present prior to thelyophilization process and persists in the resulting lyophilizedpreparation. It can be used to protect nanoparticles, liposomes, and/ormicelles during lyophilization, for example to reduce or preventaggregation, particle collapse and/or other types of damage. In anembodiment the lyoprotectant is a cryoprotectant.

In an embodiment the lyoprotectant is a carbohydrate. The term“carbohydrate,” as used herein refers to and encompassesmonosaccharides, disaccharides, oligosaccharides and polysaccharides.

In an embodiment, the lyoprotectant is a monosaccharide. The term“monosaccharide,” as used herein refers to a single carbohydrate unit(e.g., a simple sugar) that can not be hydrolyzed to simplercarbohydrate units. Exemplary monosaccharide lyoprotectants includeglucose, fructose, galactose, xylose, ribose and the like.

In an embodiment, the lyoprotectant is a disaccharide. The term“disaccharide,” as used herein refers to a compound or a chemical moietyformed by 2 monosaccharide units that are bonded together through aglycosidic linkage, for example through 1-4 linkages or 1-6 linkages. Adisaccharide may be hydrolyzed into two monosaccharides. Exemplarydisaccharide lyoprotectants include sucrose, trehalose, lactose, maltoseand the like.

In an embodiment, the lyoprotectant is an oligosaccharide. The term“oligosaccharide,” as used herein refers to a compound or a chemicalmoiety formed by 3 to about 15, preferably 3 to about 10 monosaccharideunits that are bonded together through glycosidic linkages, for examplethrough 1-4 linkages or 1-6 linkages, to form a linear, branched orcyclic structure. Exemplary oligosaccharide lyoprotectants includecyclodextrins, raffinose, melezitose, maltotriose, stachyose acarbose,and the like. An oligosaccharide can be oxidized or reduced.

In an embodiment, the lyoprotectant is a cyclic oligosaccharide. Theterm “cyclic oligosaccharide,” as used herein refers to a compound or achemical moiety formed by 3 to about 15, preferably 6, 7, 8, 9, or 10monosaccharide units that are bonded together through glycosidiclinkages, for example through 1-4 linkages or 1-6 linkages, to form acyclic structure. Exemplary cyclic oligosaccharide lyoprotectantsinclude cyclic oligosaccharides that are discrete compounds, such as αcyclodextrin, β cyclodextrin, or γ cyclodextrin.

Other exemplary cyclic oligosaccharide lyoprotectants include compoundswhich include a cyclodextrin moiety in a larger molecular structure,such as a polymer that contains a cyclic oligosaccharide moiety. Acyclic oligosaccharide can be oxidized or reduced, for example, oxidizedto dicarbonyl forms. The term “cyclodextrin moiety,” as used hereinrefers to cyclodextrin (e.g., an α, β, or γ cyclodextrin) radical thatis incorporated into, or a part of, a larger molecular structure, suchas a polymer. A cyclodextrin moiety can be bonded to one or more othermoieties directly, or through an optional linker. A cyclodextrin moietycan be oxidized or reduced, for example, oxidized to dicarbonyl forms.

Carbohydrate lyoprotectants, e.g., cyclic oligosaccharidelyoprotectants, can be derivatized carbohydrates. For example, in anembodiment, the lyoprotectant is a derivatized cyclic oligosaccharide,e.g., a derivatized cyclodextrin, e.g., 2 hydroxy propyl-β cyclodextrin,e.g., partially etherified cyclodextrins (e.g., partially etherified βcyclodextrins) disclosed in U.S. Pat. No. 6,407,079, the contents ofwhich are incorporated herein by this reference.

An exemplary lyoprotectant is a polysaccharide. The term“polysaccharide,” as used herein refers to a compound or a chemicalmoiety formed by at least 16 monosaccharide units that are bondedtogether through glycosidic linkages, for example through 1-4 linkagesor 1-6 linkages, to form a linear, branched or cyclic structure, andincludes polymers that comprise polysaccharides as part of theirbackbone structure. In backbones, the polysaccharide can be linear orcyclic. Exemplary polysaccharide lyoprotectants include glycogen,amylase, cellulose, dextran, maltodextrin and the like.

Derivatized Carbohydrate:

The term “derivatized carbohydrate,” refers to an entity which differsfrom the subject non-derivatized carbohydrate by at least one atom. Forexample, instead of the —OH present on a non-derivatized carbohydratethe derivatized carbohydrate can have —OX, wherein X is other than H.Derivatives may be obtained through chemical functionalization and/orsubstitution or through de novo synthesis—the term “derivative” impliesno process-based limitation.

Injector:

The term “injector” as used herein refers to a device that can force,e.g., propel, a fluid, e.g., a liquid, into a receiving medium.

In some of the following embodiments, various methods for generatingnanoparticles are described with reference to steps of these methods.The order in which the steps of the methods are discussed is notintended to necessarily indicate the order in which those steps must beperformed.

With reference to the flow charts of FIGS. 1 and 2, in an exemplaryembodiment of a method according to the teachings of the invention forforming polymeric nanoparticles, a polymer solution can be generated bydissolving one or more polymers, such as a polymer to which atherapeutic or an imaging agent is coupled, or with which a therapeuticor an imaging agent is associated or in which a therapeutic or animaging agent is incorporated (e.g., embedded), in a process solvent(step A). Further, an anti-solvent can be prepared (step B). While theprocess solvent is miscible, or at least partially miscible, with theanti-solvent, the polymer is not soluble in the anti-solvent in anyappreciable amount, thereby allowing a subsequent formation of polymericnanoparticles via a precipitation process (step E). For example, thesolubility of the polymer in the anti-solvent at room temperature can beless than about 0.1% by weight. Further, any additive agent(s) added tothe polymer solution is preferably not soluble in the anti-solvent inany appreciable amount. In some embodiments, the polymer solution andthe anti-solvent are filter sterilized (steps C and D) prior to theiruse in the precipitation process.

Referring to the flow chart of FIG. 2, in an exemplary embodiment forperforming the precipitation process to generate a plurality ofnanoparticles (step E in the flow chart of FIG. 1), an anti-solvent isintroduced into a static mixer, e.g., a static mixer disposed in aconduit, to create a mixed flowing stream of the anti-solvent (step 1).The polymer solution is introduced into the mixed flowing stream of theanti-solvent such that precipitation of the polymer into a plurality ofpolymeric nanoparticles occurs (step 2). The generated nanoparticles arethen separated from the anti-solvent stream, e.g., in a manner discussedin more detail below.

Without being limited to any particular theory, upon contact of thepolymer solution with the anti-solvent stream, precipitation of thepolymer into a plurality of polymeric nanoparticles occurs as a resultof rapid desolvation of the polymer. In particular, the process solventdiffuses rapidly into the anti-solvent due to its miscibility with theanti-solvent. The polymer is, however, not miscible in the anti-solventand hence aggregates (e.g., precipitates) into a plurality ofnanoparticles as the process solvent diffuses into the anti-solvent. Thestatic mixer design, the choice of the anti-solvent and the processsolvent, and in particular the mixing of the anti-solvent and theprocess solvent, can facilitate the mass transfer of solvents, therebycontrolling the size of the nanoparticles formed via precipitation. Thenanoparticles can be formed rapidly, e.g., over a milliseconds timescale. For example, in some cases the nanoparticles can form in lessthan about 10 milliseconds (e.g., in a range of about 1 millisecond toabout 10 milliseconds), or less than about 5 milliseconds (e.g., in arange of about 1 millisecond to about 5 milliseconds), subsequent to thecontact of the polymer solution with the mixed flowing stream of theanti-solvent. The rapid formation of the nanoparticles can be due tointerfacial turbulence at the interface of the solvent and theanti-solvent, which can result, e.g., from flow, diffusion and surfacetension variations.

As discussed in more detail below, the introduction of the polymersolution into a mixed flowing stream of the anti-solvent results information of polymeric nanoparticles with predictable average particlesizes and a low polydispersity index (PdI) over a wide range ofanti-solvent, and polymer solution, flow rates and average axial flowvelocities.

In some embodiments, the average particle size (Z_(ave)) can be equal toor less than about 500 nm. For example, the polymeric nanoparticles canexhibit an average particle size in a range of about 5 nm to about 500nm, or in a range of about 10 nm to about 500 nm, or in a range of about20 nm to about 500 nm, or in a range of about 30 nm to about 500 nm, orin a range of about 40 nm to about 500 nm, or in a range of about 50 nmto about 500 nm. In some embodiments, the average particle size(Z_(ave)) can be equal to or less than about 400 nm. For example, thepolymeric nanoparticles can exhibit an average particle size in a rangeof about 5 nm to about 400 nm, or in a range of about 10 nm to about 400nm, or in a range of about 20 nm to about 400 nm, or in a range of about30 nm to about 400 nm, or in a range of about 50 nm to about 400 nm. Insome embodiments, the average particle size (Z_(ave)) can be equal to orless than about 300 nm. For example, the polymeric nanoparticles canexhibit an average particle size in range of about 5 nm to about 300 nm,or in a range of about 10 nm to about 300 nm, or in a range of about 20nm to about 300 nm, or in a range of about 40 nm to about 300 nm, or ina range of about 50 nm to about 300 nm.

In some embodiments, the average particle size (Z_(ave)) of thenanoparticles can be equal to or less than about 200 nm (e.g., equal toor less than about 195 nm (and, e.g., equal to or greater than about 20nm), equal to or less than about 190 nm (and, e.g., equal to or greaterthan about 20 nm), equal to or less than about 185 nm (and, e.g., equalto or greater than about 20 nm), equal to or less than about 180 nm(and, e.g., equal to or greater than about 20 nm), equal to or less thanabout 175 nm (and, e.g., equal to or greater than about 20 nm), equal toor less than about 170 nm (and, e.g., equal to or greater than about 20nm), equal to or less than about 165 nm (and, e.g., equal to or greaterthan about 20 nm), equal to or less than about 160 nm (and, e.g., equalto or greater than about 20 nm), equal to or less than about 155 nm(and, e.g., equal to or greater than about 20 nm), equal to or less thanabout 150 nm (and, e.g., equal to or greater than about 20 nm), equal toor less than about 145 nm (and, e.g., equal to or greater than about 20nm), equal to or less than about 140 nm (and, e.g., equal to or greaterthan about 20 nm), equal to or less than about 135 nm (and, e.g., equalto or greater than about 20 nm), equal to or less than about 130 nm(and, e.g., equal to or greater than about 20 nm), equal to or less thanabout 125 nm (and, e.g., equal to or greater than about 20 nm), equal toor less than about 120 nm (and, e.g., equal to or greater than about 20nm), equal to or less than about 115 nm (and, e.g., equal to or greaterthan about 20 nm), equal to or less than about 110 nm (and, e.g., equalto or greater than about 20 nm), equal to or less than about 105 nm(and, e.g., equal to or greater than about 20 nm), equal to or less thanabout 100 nm (and, e.g., equal to or greater than about 20 nm), equal toor less than about 95 nm (and, e.g., equal to or greater than about 20nm), equal to or less than about 90 nm (and, e.g., equal to or greaterthan about 20 nm), equal to or less than about 85 nm (and, e.g., equalto or greater than about 20 nm), equal to or less than about 80 nm (and,e.g., equal to or greater than about 20 nm), equal to or less than about75 nm (and, e.g., equal to or greater than about 20 nm), equal to orless than about 70 nm (and, e.g., equal to or greater than about 20 nm),equal to or less than about 65 nm (and, e.g., equal to or greater thanabout 20 nm), equal to or less than about 60 nm (and, e.g., equal to orgreater than about 20 nm), equal to or less than about 55 nm or 50 nm(and, e.g., equal to or greater than about 20 nm)). For example, theaverage particle size can be in a range of about 50 nm to about 200 nm,or in a range of about 100 nm to about 200 nm.

In general, a wide range of anti-solvent and polymer solution flow ratescan be employed. By way of example, the flow rate of the anti-solventstream can be in a range of about 20 ml/min to about 2000 ml/min, andthe flow rate of the polymer solution can be in a range of about 4ml/min to about 200 ml/min, for example, in a range of about 5 ml/min toabout 100 ml/min. In some embodiments, the flow rate of the anti-solventstream is substantially greater than the flow rate of the polymersolution into the anti-solvent stream. For example, the flow rate of theanti-solvent stream can be at least about 2 times, or at least about 3times, at least about 5 times, or at least about 10 times, greater thanthe flow rate of the polymer solution. In some other embodiments, theanti-solvent flow rate and the polymer solution flow rate can have a 1:1ratio.

In some embodiments, the concentration of the polymer solution and/orthe concentration of the anti-solvent can be changed so as to adjust theaverage particle size of the nanoparticles.

In many embodiments, the static mixer generates a mixed flowing streamof the anti-solvent that presents a substantially isotropic mixedanti-solvent environment to the incoming polymer solution, thus ensuringthat the formed nanoparticles will exhibit a low polydispersity index.For example, the static mixer can create sufficient radial and/ortangential motion of the anti-solvent to rapidly create a substantiallyuniform mixed environment, thus facilitating formation of nanoparticleswith a low polydispersity index. For example, as indicated above, thepolydispersity index can be equal to or less than about 0.25, e.g., in arange of about 0.05 to about 0.1. By way of example, in someembodiments, the static mixer generates a mixed anti-solvent environmentthat is substantially isotropic over at least about 50%, or at leastabout 60%, or at least about 70%, or at least about 90%, or 100% of thevolume of the conduit in which the static mixer is disposed.

Further, a mixed flowing stream of the anti-solvent can allow theintroduction of the polymer solution at a variety of velocities (andcorresponding momentum values) into the anti-solvent stream. Even at alow momentum, the polymer solution will encounter a highly mixedanti-solvent environment, which will lead to formation of nanoparticleswith predictable average particle size and polydispersity index.

In some embodiments, the polymer solution can be introduced into themixed flowing stream of the anti-solvent at an intermediate location ofthe static mixer. Alternatively, the polymer solution can be introducedinto the anti-solvent flowing stream in proximity to a proximal or adistal end of the mixer. In some embodiments, the polymer solution isintroduced into the mixed flowing stream of the anti-solvent at anon-zero angle, e.g., an acute angle, relative to the anti-solvent flowdirection. For example, the polymer solution stream can intersect theanti-solvent flowing stream at an angle in a range of about 50 degreesto about 90 degrees. In some embodiments, the polymer solution isinjected into the mixed flowing stream of the anti-solvent either at anangle relative to the direction of the anti-solvent flow orsubstantially parallel to the direction of the anti-solvent flow.

In some embodiments, rather that utilizing a single static mixer unit,multiple static mixer units can be employed to provide a mixed flowingstream of an anti-solvent within a selected portion of a conduit. Insome such embodiments, the static mixer units are oriented in astaggered configuration relative to one another so as to enhance themixing of the anti-solvent.

A variety of polymers, process solvents, and anti-solvents can beemployed in the precipitation process to form nanoparticles. By way ofexample, the polymers can include the following monomers (or sub-units):acrylates, acrylonitriles such as methacrylnitrile, vinyls, aminoalkyls,styrenes, and lactic acids. Some examples of acrylates include, withoutlimitation, methyl acrylate, ethyl acrylate, propyl acrylate, n-butylacrylate, isobutyl acrylate, 2-ethyl acrylate, and t-butyl acrylate.Some examples of vinyls include, without limitation, vinyl acetate,vinylversatate, vinylpropionate, vinylformamide, vinylacetamide,vinylpyridines and vinylimidazole. Some examples of aminoalkyls include,without limitation, aminoalkylacrylates, aminoalkylmethacrylates andaminoalkyl(meth)acrylamides.

In some embodiments, the polymer can be an amphiphilic copolymer that isformed of monomers exhibiting different hydrophilic and hydrophobicproperties. For example, in some embodiments, the polymer has ahydrophilic portion and a hydrophobic portion. In some embodiments, thepolymer is a block copolymer. In some embodiments, the amphiphiliccopolymer is formed of blocks (groups) of monomers or sub-units, wheresome blocks are substantially hydrophobic while other blocks aresubstantially hydrophilic. For example, in diblock copolymers the blocksare arranged as a series of two blocks having similar hydrophobic orhydrophilic properties while in triblock copolymers, the blocks arearranged as a series of three blocks having similar hydrophobic orhydrophilic properties. In some embodiments, the amphiphilic polymercomprises two regions, one of which is hydrophilic and the otherhydrophobic, where the two regions together comprise at least about 70%by weight of the polymer (e.g., at least about 80%, at least about 90%,at least about 95%).

In some embodiments, the hydrophobic portion of the polymer is abiodegradable polymer (e.g., PLA, PGA, PLGA, PCL, PDO, polyanhydrides,polyorthoesters, or chitosan). In some embodiments, the hydrophobicportion of the polymer is PLA. In some embodiments, the hydrophobicportion of the polymer is PGA. In some embodiments, the hydrophobicportion of the polymer is a copolymer of lactic and glycolic acid (e.g.,PLGA).

In some embodiments, the hydrophilic portion of the polymer ispolyethylene glycol (PEG). In some embodiments, the hydrophilic portionof the polymer has a molecular weight of from about 1 kDa to about 20kDa (e.g., from about 1 kDa to about 15 kDa, from about 2 kDa to about12 kDa, from about 6 kDa to about 20 kDa, from about 5 kDa to about 10kDa, from about 7 kDa to about 10 kDa, from about 5 kDa to about 7 kDa,from about 6 kDa to about 8 kDa, about 6 kDa, about 7 kDa, about 8 kDa,or about 9 kDa). In some embodiments, the ratio of molecular weight ofthe hydrophilic to hydrophobic portions of the polymer is from about1:20 to about 1:1 (e.g., about 1:10 to about 1:1, about 1:2 to about1:1, or about 1:6 to about 1:3).

In some embodiments, the hydrophilic portion of the polymer terminatesin a hydroxyl moiety prior to conjugation to an agent. In someembodiments, the hydrophilic portion of the polymer terminates in analkoxy moiety. In some embodiments, the hydrophilic portion of thepolymer is a methoxy PEG (e.g., a terminal methoxy PEG).

In some embodiments, the hydrophilic portion of the polymer is attachedto the hydrophobic portion through a covalent bond. In some embodiments,the hydrophilic polymer is attached to the hydrophobic polymer throughan amide, ester, ether, amino, carbamate, or carbonate bond (e.g., anester or an amide).

In some embodiments, the polymer is a biodegradable polymer (e.g.,polylactic acid (PLA), polyglycolic acid (PGA), poly(lactic-co-glycolicacid) (PLGA), polycaprolactone (PCL), polydioxanone (PDO),polyanhydrides, polyorthoesters, or chitosan). In some embodiments, thepolymer is a hydrophobic polymer. In some embodiments, the polymer isPLA. In some embodiments, the polymer is PGA.

In some embodiments, the polymer is a copolymer of lactic and glycolicacid (poly(lactic-co-glycolic acid) (PLGA)). In some embodiments, thepolymer is a PLGA-ester. In some embodiments, the polymer is aPLGA-lauryl ester. In some embodiments, the polymer comprises a terminalfree acid prior to conjugation to an agent. In some embodiments, thepolymer comprises a terminal acyl group (e.g., an acetyl group). In someembodiments, the ratio of lactic acid monomers to glycolic acid monomersis from about 0.1:99.9 to about 99.9:0.1. In some embodiments, the ratioof lactic acid monomers to glycolic acid monomers is from about 75:25 toabout 25:75 (e.g., about 50:50 or about 75:25).

In some embodiments, the average molecular weight of the polymer is fromabout 1 kDa to about 20 kDa (e.g., from about 1 kDa to about 15 kDa,from about 2 kDa to about 12 kDa, from about 6 kDa to about 20 kDa, fromabout 5 kDa to about 10 kDa, from about 7 kDa to about 10 kDa, fromabout 5 kDa to about 7 kDa, from about 6 kDa to about 8 kDa, about 6kDa, about 7 kDa, about 8 kDa, or about 9 kDa). In some embodiments, thepolymer has a glass transition temperature of about 20° C. to about 60°C. In some embodiments, the polymer has a polymer polydispersity indexequal to or less than about 2.5 (e.g., less than or equal to about 2.2,or less than or equal to about 2.0).

By way of further illustration, some examples of suitable polymersinclude poly(lactide-co-glycolide), poly(lactide),poly(epsilon-caprolactone), poly(isobutylcyanoacrylate),poly(isohexylcyanoacrylate), poly(n-butylcyanoacrylate), poly(acrylate),poly(methacrylate), poly(lactide)-poly(ethylene glycol),poly(lactide-co-glycolide)-poly(ethylene glycol),poly(epsilon-caprolactone)-poly(ethylene glycol), andpoly(hexadecylcyanoacrylate-co-poly(ethylene glycol) cyanoacrylate).

In some embodiments, the polymer can include one or more graftedmoieties, e.g., alkyl chains of 4 to 18 carbons, such as a grafted butylgroup. In some embodiments, such grafted moieties can enhance thesalvation of the polymer in the process solvent and/or the stability ofthe polymeric nanoparticles formed in the subsequent steps.

In some embodiments, a single agent is attached to a single polymer,e.g., to a terminal end of the polymer. In some embodiments, a pluralityof agents are attached to a single polymer (e.g., 2, 3, 4, 5, 6, ormore). In some embodiments, the agents are the same agent. In someembodiments, the agents are different agents. In some embodiments, theagent is a therapeutic agent or an imaging agent.

In an embodiment, the agent is poorly soluble in water, e.g., it has asolubility of less than about 1 mg/liter, or about 0.9 mg/liter, orabout 0.8 mg/liter, or about 0.7 mg/liter, or about 0.6 mg/liter, orabout 0.5 mg/liter in unbuffered water (pH of 7)_. In an embodiment, theagent has a molecular weight of between about 200 to 1500, 400 to 1500,200 to 1000, 400 to 1000, 200 to 800, or 400 to 800 Daltons.

In some embodiments, the therapeutic agent is an anti-neoplastic agent.In some embodiments, the anti-neoplastic agent is an alkylating agent, avascular disrupting agent, a microtubule targeting agent, a mitoticinhibitor, a topoisomerase inhibitor, an anti-angiogenic agent or ananti-metabolite. In some embodiments, the anti-neoplastic agent is ataxane (e.g., paclitaxel, docetaxel, larotaxel or cabazitaxel). In someembodiments, the anti-neoplastic agent is an anthracycline (e.g.,doxorubicin). In some embodiments, the anti-neoplastic agent is anepothilone (e.g., ixabepilone, epothilone B, epothilone D, BMS310705,dehydelone or ZK-epothilone). In some embodiments, the anti-neoplasticagent is a platinum-based agent (e.g., cisplatin). In some embodiments,the anti-neoplastic agent is a pyrimidine analog (e.g., gemcitabine,premetrexed, floxuridine, fluorouracil (5-FU)).

In some embodiments, the anti-neoplastic agent is paclitaxel, attachedto the polymer through the 2′ or 7 carbon position, or both the 2′ and 7carbon positions. In some embodiments, the agent is linked to thepolymer through the 7 position and has an acyl group at the 2′ position(e.g., wherein the agent is a taxane such as paclitaxel, docetaxel,larotaxel or cabazitaxel).

In some embodiments, the anti-neoplastic agent is docetaxel. In someembodiments, the anti-neoplastic agent is docetaxel-succinate. In someembodiments, the anti-neoplastic agent is doxorubicin. In someembodiments, the anti-neoplastic agent is larotaxel. In someembodiments, the anti-neoplastic agent is cabazitaxel.

In some embodiments, the therapeutic agent is an agent for the treatmentor prevention of cardiovascular disease. In some embodiments, thetherapeutic agent is an agent for the treatment or prevention of aninflammatory or autoimmune disease.

In some embodiments, the agent is attached directly to the polymer,e.g., through a covalent bond. In some embodiments, the agent isattached to a terminal end of the polymer via an amide, ester, ether,amino, carbamate or carbonate bond. In some embodiments, the agent isattached to a terminal end of the polymer. In some embodiments, thepolymer comprises one or more side chains and the agent is directlyattached to the polymer through one or more of the side chains.

In some embodiments, a single agent is attached to a polymer. In someembodiments, multiple agents are attached to a polymer (e.g., 2, 3, 4 ormore agents). In some embodiments, the agents are the same agent. Insome embodiments, the agents are different agents.

In some embodiments, the agent is doxorubicin, and is covalentlyattached to the polymer through, e.g., an amide bond.

In some embodiments, the polymer-agent conjugate is:

wherein about 40% to about 60% of R substituents are hydrogen (e.g.,about 50%) and about 40% to about 60% are methyl (e.g., about 50%); andwherein n is an integer from about 90 to about 170 (e.g., n is aninteger such that the molecular weight of the polymer-agent conjugate isfrom about 6 kDa to about 11 kDa).

In some embodiments, the agent is paclitaxel, and is covalently attachedto the polymer through, e.g., an ester bond.

In some embodiments, the polymer-agent conjugate is:

wherein about 40% to about 60% of R substituents are hydrogen (e.g.,about 50%) and about 40% to about 60% are methyl (e.g., about 50%); andwherein n is an integer from about 90 to about 170 (e.g., n is aninteger such that the molecular weight of the polymer-agent conjugate isfrom about 6 kDa to about 11 kDa).

In some embodiments, the polymer-agent conjugate is:

wherein about 40% to about 60% of R substituents are hydrogen (e.g.,about 50%) and about 40% to about 60% are methyl (e.g., about 50%); andwherein n is an integer from about 90 to about 170 (e.g., n is aninteger such that the molecular weight of the polymer-agent conjugate isfrom about 6 kDa to about 11 kDa).

In some embodiments, the paclitaxel is attached through both the 2′ andthe 7 carbons. In some embodiments, the polymer-agent is provided as amixture containing one or more or all of, drug-polymer species coupledthrough the 2′ carbon, drug-polymer species coupled through the 7carbon, and drug-polymer species coupled through both the 2′ and the 7carbons.

In some embodiments, the agent is paclitaxel, and is covalently attachedto the polymer via a carbonate bond.

In some embodiments, the agent is docetaxel, and is covalently attachedto the polymer through, e.g., an ester bond.

In some embodiments, the polymer-agent conjugate is:

wherein about 40% to about 60% of R substituents are hydrogen (e.g.,about 50%) and about 40% to about 60% are methyl (e.g., about 50%); andwherein n is an integer from about 90 to about 170 (e.g., n is aninteger such that the molecular weight of the polymer-agent conjugate isfrom about 6 kDa to about 11 kDa).

In some embodiments, the polymer-agent conjugate is:

wherein about 40% to about 60% of R substituents are hydrogen (e.g.,about 50%) and about 40% to about 60% are methyl (e.g., about 50%); andwherein n is an integer from about 90 to about 170 (e.g., n is aninteger such that the molecular weight of the polymer-agent conjugate isfrom about 6 kDa to about 11 kDa).

In some embodiments, the docetaxel is attached through both the 2′ andthe 7 carbons. In some embodiments, the polymer-agent is provided as amixture containing one or more or all of, drug-polymer species coupledthrough the 2′ carbon, drug-polymer species coupled through the 7carbon, and drug-polymer species coupled through both the 2′ and the 7carbons.

In some embodiments, the agent is docetaxel, and is covalently attachedto the polymer through a carbonate bond.

In some embodiments, the agent is attached to the polymer through alinker. In some embodiments, the linker is an alkanoate linker. In someembodiments, the linker is a PEG-based linker. In some embodiments, thelinker comprises a disulfide bond. In some embodiments, the linker is aself-immolative linker. In some embodiments, the linker is an amino acidor a peptide (e.g., glutamic acid, branched glutamic acid orpolyglutamic acid).

In some embodiments the linker is a multifunctional linker. In someembodiments, the multifunctional linker has 2, 3, 4 or more reactivemoieties that may be functionalized with an agent. In some embodiments,all reactive moieties are functionalized with an agent. In someembodiments, not all of the reactive moieties are functionalized with anagent (e.g., the multifunctional linker has four reactive moieties, andonly one, two or three react with an agent.)

In some embodiments, the polymer-agent conjugate is:

wherein about 40% to about 60% of R substituents are hydrogen (e.g.,about 50%) and about 40% to about 60% are methyl (e.g., about 50%); andwherein n is an integer from about 90 to about 170 (e.g., n is aninteger such that the molecular weight of the polymer-agent conjugate isfrom about 6 kDa to about 11 kDa).

In some embodiments, two agents are attached to a polymer via amultifunctional linker. In some embodiments, the two agents are the sameagent. In some embodiments, the two agents are different agents. In someembodiments, the agent is docetaxel, and is covalently attached to thepolymer via a glutamate linker.

In some embodiments, the polymer-agent conjugate is:

wherein about 40% to about 60% of R substituents are hydrogen (e.g.,about 50%) and about 40% to about 60% are methyl (e.g., about 50%); andwherein n is an integer from about 90 to about 170 (e.g., n is aninteger such that the molecular weight of the polymer-agent conjugate isfrom about 6 kDa to about 11 kDa).

In some embodiments, four agents are attached to a polymer via amultifunctional linker. In some embodiments, the four agents are thesame agent. In some embodiments, the four agents are different agents.In some embodiments, the agent is docetaxel, and is covalently attachedto the polymer via a bis(glutamate) linker.

In some embodiments, the polymer-agent conjugate is:

wherein 2′-docetaxel is:

wherein about 40% to about 60% of R substituents are hydrogen (e.g.,about 50%) and about 40% to about 60% are methyl (e.g., about 50%); andwherein n is an integer from about 90 to about 170 (e.g., n is aninteger such that the molecular weight of the polymer-agent conjugate isfrom about 6 kDa to about 11 kDa).

In some embodiments, the polymer, e.g., the hydrophilic portion of anamphiphilic copolymer, comprises a terminal conjugate. In someembodiments, the terminal conjugate is a targeting agent or a dye. Insome embodiments, the terminal conjugate is a folate or a rhodamine. Insome embodiments, the terminal conjugate is a targeting peptide (e.g.,an RGD peptide). By way of example, the targeting agent can becovalently linked to the polymer. In some embodiments, the targetingagent can be capable of binding to, or otherwise associating with, atarget biological entity, e.g., a membrane component, a cell surfacereceptor, a prostate specific membrane antigen, or the like. In someembodiments, the targeting agent can cause the nanoparticlesadministered to a subject to become localized to a tumor, a diseasesite, a tissue, an organ, a type of cell, e.g., a cancer cell. In someembodiments, the targeting agent can be selected from the group ofnucleic acid aptamers, growth factors, hormones, cytokines,interleukins, antibodies, integrins, fibronectin receptors,p-glycoprotein receptors, peptides and cell binding sequences.

In some embodiments, a radiopharmaceutical agent e.g., aradiotherapeutic agent, a radioimaging agent, or other radioisotope canbe coupled to, associated with or incorporated in the polymer, e.g.,embedded in the polymer.

In some embodiments, the process solvent is an organic solvent (or amixture of two or more organic solvents). In some embodiments, theprocess solvent is capable of dissolving at least about 0.1%, or atleast about 0.2%, by weight of the polymer at room temperature.

Some examples of suitable process solvents include, without limitation,acetone, ether, alcohol, tetrahydrofuran, 2-pyrrolidone,N-Methyl-2-pyrrolidone (NMP), dimethylformamide (DMF), dimethylacetamide(DMA), methyl acetate, ethyl formate, methyl ethyl ketone (MEK), methylisobutyl ketone (MIBK), methyl propyl ketone, isopropyl ketone,isopropyl acetate, acetonitrile (MeCN) and dimethyl sulfoxide (DMSO).

In some embodiments, the anti-solvent can be an aqueous (water-based)solution, another solvent, a combination of a solvent and an aqueoussolution, or a combination of one or more organic solvents. In someembodiments, the anti-solvent can be purified water. Some other examplesof suitable anti-solvents include, without limitation, methanol,ethanol, n-propanol, isopropanol, n-butanol, ethyl ether, andwater:ethanol (e.g., 50:50). In some cases, the anti-solvent can be aliquefied gas, such as carbon dioxide under adequate pressure.

In some embodiments, the anti-solvent can include a colloid stabilizer,e.g., to inhibit aggregation of the formed nanoparticles. Some examplesof suitable colloid stabilizers include, without limitation, poly(vinylalcohol) (PVA), Dextran and pluronic F68, poly (vinyl pyrrolidone),solutol, Tween 80, poloxamer, carbopol, poly-ethylene glycol (PEG),sodium dodecyl sulfate, poly (ε-caprolactone), peptides, andcarbohydrates. Another example of a colloid stabilizers includes,without limitation, a PEG-lipid (e.g., PEG-ceramide, d-alpha-tocopherylpolyethylene glycol 1000 succinate,1,2-Distearoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)] or lecithin). Insome embodiments, the PVA is from about 5 to about 45 kDa, for example,the PVA is from about 5 to about 30 kDa (e.g., the PVA is from about 5to about 20 kDa), and up to about 98% hydrolyzed (e.g., about 85%hydrolyzed). In some embodiments, the viscosity of the PVA (4% PVA inwater), measured by utilizing the falling ball method, is in a range ofabout 2.5 to about 6.5 mPasec (e.g., in a range of about 2.5 to about3.5 mPasec at a temperature of about 20° C.). In some embodiments, theviscosity of the PVA (4% PVA in water), measured by utilizing thefalling ball method, is in a range of about 3.4 to about 4.6 mPasec.

In some embodiments, the polymer solution can have one or more additivemolecules. As discussed above, in some embodiments, the additivemolecules are embedded in the polymer prior to formation of the polymersolution. In other embodiments, the additive molecules can becomeembedded in the polymeric nanoparticles during the precipitationprocess. For example, in some embodiments, the additive molecule can beconjugated to the polymer, e.g., via covalent bonding, and theconjugated polymer can be dissolved in a process solvent to form thepolymer solution. In other cases, the additive molecules can be presentin the polymer solution without being conjugated to the polymer andbecome subsequently trapped in polymeric nanoparticles during theprecipitation process.

By way of example, the additive molecules can be a therapeutic, or animaging agent or a combination of therapeutic and imaging agents. Someexamples of suitable therapeutic agents include, without limitation,anti-neoplastic agents, anti-inflammatory agents, cardiovascular activeagents, and anti-metabolites.

In some embodiments, the imaging agent can be coupled, e.g., conjugatedto the polymer, for incorporation in the nanoparticles. In otherembodiments, the imaging agent can be coupled, e.g., via a chelatingagent, to a therapeutic agent, which is in turn coupled, e.g.,conjugated, to the polymer. The imaging agents can include, e.g.,radioactive, non-radioactive, or fluorescent labels. Some examples ofimaging agents include, without limitation, radiopharmaceuticals such asTechnetium Bicisate, Ioxaglate, and Fluorodeoxyglucose, label-free Ramanimaging agents, encapsulate MRI contrast agent Gd-DTPA, and rhodamine 6Gas a fluorescent agent. In some embodiments, the imaging agent can beradiolabeled docetaxel (e.g., 3H-radiolabeled or 14C-radiolabeleddocetaxel), or radiolabeled paclitaxel.

Referring again to the flow chart of FIG. 1, the nanoparticles formed inthe precipitation process can be collected, e.g., as a suspension (stepF). For example, the formed nanoparticles entrained in a mixture ofanti-solvent and process solvent (in many cases, mostly anti-solvent)flowing downstream from the static mixer can be introduced into a tank,e.g., a tank containing a liquid, e.g., deionized water. A suspension ofthe nanoparticles can then be collected from the tank.

The collected nanoparticles can be subjected to a variety of processes,which may be conducted aseptically, to yield aqueous or non-aqueoussolutions, dispersions or powders. For example, as discussed in moredetail below, a concentrated suspension of the nanoparticles can belyophilized to yield a powder containing the nanoparticles, e.g., asterile powder. In some applications, such a sterile powder of thenanoparticles can be subsequently reconstituted into sterile injectiblesolutions or dispersions.

For example, with continued reference to the flow chart of FIG. 1, thecollected suspension containing the nanoparticles can be diafiltered andconcentrated (step G). By way of example, the suspension containing thenanoparticles can be diafiltered, e.g., to remove at least a portion ofthe process solvent, the colloid stabilizer or other additives added tothe anti-solvent. In some embodiments, the diafiltration (also known inthe art as crossflow filtration) can be performed in multiple steps. Insome such embodiments, the nanoparticles can be washed, e.g., by usingdeionized water, between successive diafilteration steps. Further, insome embodiments the diafilteration is conducted preferably in acontinuous fashion, i.e., by adding wash solution during thediafiltration process.

In some embodiments in which the polymeric nanoparticles includetherapeutic and/or imaging agents, the concentration of these agents canbe monitored (step H) while the suspension containing the nanoparticlesis being concentrated. While in some cases such monitoring is performedcontinuously, in other cases it can be performed at multiple discretetimes. By way of example, high pressure liquid chromotagraphy (HPLC) canbe employed to assay the suspension as the volume of the anti-solventand process solvent mixture is reduced.

With continued reference to the flow chart of FIG. 1, in this exemplaryembodiment, a lyoprotectant is added to the concentrated suspension ofthe nanoparticles to protect the nanoparticles from damage and/or toretard permanent aggregation of the nanoparticles when subsequentlysubjected to lyophilization. The lyoprotectant can also facilitate theresuspension of the nanoparticles. Some examples of suitablelyoprotectants include, without limitation, a derivatized cyclicoligosaccharide, e.g., a derivatized cyclodextrin, e.g., 2 hydroxypropyl-β cyclodextrin, e.g., partially etherified cyclodextrins (e.g.,partially etherified β cyclodextrins) disclosed in U.S. Pat. No.6,407,079, the contents of which are incorporated herein by thisreference.

In step (J), the concentrated suspension containing the nanoparticlesand the lyoprotectant can then be stored in one or more suitablevessels, e.g., vials, and lyophilized in a manner known in the art (stepK). The vials can then be sealed to protect the nanoparticles fromspoilage. By way of example, the lyophilization can be achieved byinitially freezing the concentrated suspension followed by a primarydrying phase in which the ambient pressure to which the concentratedsuspension is subjected is lowered (e.g. to a few millibars) whilesupplying enough heat to cause sublimation of frozen liquid, mostlyfrozen water in many implementations at this stage. In a secondarydrying phase, unfrozen liquid (e.g., water molecules), if any, can beremoved by raising the temperature above that in the primary. In someembodiments, upon completion of the freeze-drying process, an inert gas,such as nitrogen, can be introduced into the vessel containing thelyophilized nanoparticles prior to sealing the vessel.

As discussed above, the use of a static mixer in generating a mixedflowing stream of an anti-solvent into which a polymer solution isintroduced for generating nanoparticles advantageously allows operatingthe precipitation process at a variety of anti-solvent, and polymersolution, flow rates. It has been discovered that the anti-solvent flowrate and/or the polymer solution flow rate can be adjusted to controlthe average particle size of the formed nanoparticles in a predictablemanner. Hence, in another aspect, the invention provides a method forcontrolling particle size of nanoparticles formed by precipitation.

For example, with reference to the flow chart of FIG. 3, in such amethod, an anti-solvent flow is introduced into a static mixer togenerate a mixed flowing stream of the anti-solvent (step 1). A polymersolution is then introduced into the mixed flowing stream of theanti-solvent so as to provide a plurality of nanoparticles byprecipitation (step 2). The flow rate of the anti-solvent through thestatic mixer is controlled (step 3) so as to adjust an average particlesize of the nanoparticles.

By way of example, the flow rate of the anti-solvent through the staticmixer can be changed in a range of about 20 ml/min to about 2000 ml/minso as to vary the average particle size in a range of about 50 nm toabout 200 nm.

In some embodiments the flow rate of the anti-solvent is changed, whilemaintaining the flow rate of the polymer solution substantiallyconstant, so as to adjust the average particle size of thenanoparticles. For example, in some embodiments that utilize high ratiosof the flow rate of the anti-solvent relative to that of the polymersolution, e.g., ratios of 10:1 or higher, the average particle size canbe controlled by adjusting only the anti-solvent flow rate. In otherembodiments, the polymer solution flow rate is changed, whilemaintaining the flow rate of the anti-solvent substantially constant, soas to adjust the average particle size of the nanoparticles.Alternatively, both the anti-solvent flow rate and that of the polymersolution can be concurrently changed so as to adjust the averageparticle size of the nanoparticles.

In some embodiments, the concentration of the polymer in the polymersolution and/or the concentration of the colloid stabilizer added to theanti-solvent can be changed so as to adjust the average particle size ofthe nanoparticles.

The above processes for generating nanoparticles with controlled averageparticle sizes and a low polydispersity index find a variety ofapplications, such as nanopharmaceutical therapeutic applications. Forexample, they can be employed to fabricate nano-scale drug deliverysystems for selectively targeting disease tissue. For example, polymericnanoparticles embedded with therapeutic agents can be formed forselectively targeting diseases and disorders such as oncologicaldiseases, autoimmune diseases as well as cardiovascular diseases.

The ability to predictably control the average particle size andparticle size distribution afforded by the methods of the inventionallows optimizing selective delivery of such therapeutic nanoparticles.For example, the average particle size of the nanoparticles can beselected to allow their preferential accumulation in cancerous tumorsvia their passage through leaky blood vessels of such tumors. Further, anarrow size distribution of the nanoparticles can be employed to ensurethat the therapeutic nanoparticles can effectively target canceroustumors.

Further, in some embodiments, polymeric nanoparticles can be employedfor sustained drug delivery. For example, in some embodiments, atherapeutic agent can be entrapped within a nanoparticle formed of abiodegradable polymer. As the polymer coating is degraded, the entrappedagent can be released into a subject to whom the nanoparticles have beendelivered.

In addition, the nanoparticles can be formed so as to be masked from ahost's immune system, thereby exhibiting reduced immunogenicity andantigenecity. By way of example, the polymer nanoparticles can bePEGylated to extend their circulation time in a host. PEGylatednanoparticles can evade a subject's immune system, and PEGylatedchemotherapeutic nanoparticles can lower the toxicity of the therapeuticagent and reduce unwanted side effects.

In some embodiments, the above processes for generating polymericnanoparticles can allow controlling the amount of a therapeutic or animaging agent that can be loaded onto a nanoparticle (e.g., coupled to,associated with or incorporated into the nanoparticles), and controllingthe release rate of such an agent upon introduction of the nanoparticleinto a subject. For example, the amount of a therapeutic agent that canbe loaded onto a nanoparticle can vary based on the degree of branchingexhibited by a linker attached to the polymer from which thenanoparticle is formed and to which the agent can be coupled.

The applications of nanoparticles, and particularly polymericnanoparticles, formed in accordance with the teachings of the inventionare not limited to therapeutic applications. For example, thenanoparticles can also be employed in imaging applications.

The polymeric nanoparticles can be delivered to a subject in a varietyof ways. For example, the nanoparticles can be combined with suitablesupplemental additives, such as water, ethanol, propyleneglycol,polyethyleneglycol, glycerol, vegetable oils, and ethyloleate, forparenteral injection into a subject. In some embodiments, thenanoparticles can be administered orally, e.g., via encapsulation oflyophilized nanoparticles using known excipients. In some embodiments,the nanoparticles can be administered by inhalation, e.g., vianebulization, propellant or a dry powder device. In some embodiments,the nanoparticles can be administered mucosally (e.g., via vaginal orrectal mucosa). In some embodiments, the nanoparticles can be applied ina topical form to a subject's tissue. In some embodiments, thenanoparticles can be administered ophthalmically.

FIG. 4A schematically depicts a device 40 according to an embodiment ofthe invention for generating polymeric nanoparticles. The device 40includes a conduit 42, e.g., a hollow tube, that extends axially from afirst input (inlet) port 44, through which a fluid, e.g., ananti-solvent, can be introduced into the conduit, and an output (outlet)port 46. A static mixer 48 is disposed in the conduit to receive thefluid entering the conduit through the input port 44. The exemplarystatic mixer 48 extends from a proximal end (PE) to a distal end (DE),and includes a plurality of stationary baffles 48 a that cause mixing ofthe fluid as it flows through the mixer. Different types of staticmixers can be employed in the device 40. By way of example, somesuitable static mixers are disclosed below in the Examples section. Byway of further examples, static mixers disclosed in U.S. Pat. Nos.3,286,992 and 4,511,258 entitled, respectively, “Mixing Device,” and“Static Material Mixing Apparatus,” which are herein incorporated byreference in their entirety, can be employed. By way of another example,in some embodiments, static mixers marketed by Chemineer, Inc. of Ohio,U.S.A. under the trade designation Kenics static mixers can be employed.

The device 40 further includes a second input port 50 through which asecond fluid (e.g., another fluid such as a polymer solution asdiscussed below) can be introduced into the fluid flowing axially alongthe conduit 42 through the static mixer 48. In this embodiment, thesecond input port 50 is disposed at an intermediate location between theproximal end (PE) and the distal end (DE) of the static mixer 48.

While in this embodiment the second port is configured to introduce astream of fluid, e.g., a polymer solution, into the fluid, e.g.,anti-solvent, flowing axially through the conduit at an angle of about90 degrees relative to the axial flow direction, in other embodimentsthe second port can be configured such that the direction of the flow ofthe second fluid would intersect the axial flow direction at an angleother than 90 degrees.

By way of example, FIG. 4B schematically depicts another embodiment of adevice in which the second input port 50 is configured to introduce afluid into the conduit along a direction that forms a non-orthogonalangle with the direction of axial flow.

In use, an anti-solvent flowing stream is established through theconduit 42 via the input port 44, e.g., causing the anti-solvent to flowfrom a reservoir (not shown) into the conduit, for example, via pumping,as discussed in more detail below. The static mixer causes mixing of theflowing anti-solvent so as to provide a mixed flowing stream of theanti-solvent before the stream reaches the second input port 50. Once amixed flowing stream of the anti-solvent has been established, a polymersolution can be introduced into the anti-solvent stream via the secondinput port 50 (e.g., by causing the polymer solution to flow from areservoir (not shown) into the conduit, for example, via pumping, asdiscussed in more detail below).

As discussed above, the contact of the polymer solution with theanti-solvent results in precipitation of the polymer into a plurality ofpolymeric nanoparticles that are carried by the stream of theanti-solvent away from the static mixer. The formed nanoparticles canthen be collected as a suspension in a mixture of anti-solvent and theprocess solvent. As discussed above, in many embodiments, the rate offlow of the anti-solvent through the conduit 42 is substantially greaterthan the flow rate of the polymer solution into the conduit, e.g., by afactor of about 10 or more. Hence, in such embodiments, thenanoparticles are surrounded primarily by the anti-solvent—including anyadditive(s) such as a colloid stabilizer added to the anti-solvent—asthey move down the conduit to a collection device—though the collectiondevice receives typically the process solvent and, in some casesadditives added to the process solvent, as well. Further, in manyembodiments, the flow rate of the anti-solvent is sufficiently fast toensure that the polymer solution entering the conduit would interactwith a fresh batch of anti-solvent that is substantially free of processsolvent and polymeric material that had previously entered the conduit.

While in the above device 40 the second input port is positioned at anintermediate location relative to proximal and distal ends of the staticmixer, in other embodiments the second input port can be positioned inproximity to the proximal end or the distal end of the static mixer. Byway of example, FIG. 5A schematically depicts a device 52 according toan alternative implementation of the above device 40 for generatingnanoparticles in which the second input port 50 is positioned inproximity to the distal end (DE) of the static mixer. For example, theinput port 50 can be offset from the distal end of the mixer by one ortwo mixing elements.

By way of another example, FIG. 5B schematically depicts a device 54according to another alternative implementation of the above device 40in which the second input port 50 is positioned in proximity to theproximal end (PE) of the static mixer, e.g., offset by one or two mixingelements from the proximal end. In some embodiments, the static mixer ispreferably selected to generate a mixed flowing stream of theanti-solvent over a short length of the mixer. In some embodiments thestatic mixer can be chosen such that its mixing effect on theanti-solvent flow would even be present slightly upstream from theproximal end of the mixer. In such embodiments, as shown schematicallyin FIG. 5C, the second input port can be positioned slightly upstreamfrom the static mixer but sufficiently close to its proximal end suchthat the incoming polymer solution would interact with a mixed flowingstream of the anti-solvent.

The static mixer can also be chosen such that its mixing effect on theanti-solvent flow is present downstream from the distal end of themixer. In such embodiments, as shown schematically in FIG. 5D, thesecond input port 50 of a device 58′ can be positioned slightlydownstream from the static mixer 48 but sufficiently close to its distalend DE (e.g., within approximately 1-2 mixing element lengths) such thatthe incoming polymer solution would interact with a mixed flowing streamof the anti-solvent.

In some embodiments, a plurality of input ports for introducing thepolymer solution into a mixed flowing stream of anti-solvent can beprovided. For example, as shown in FIG. 5E, a device 58″ can include aplurality of polymer solution input ports 50 a, 50 b on opposite sidesof the conduit 42. In other embodiments, the plurality of polymersolution input ports can be located on the same side of the conduitand/or can be spaced at various intervals from each other along thelength of the conduit 42.

In other embodiments, rather than utilizing a single static mixer unit,a plurality of static mixer units can be employed. For example, FIG. 6Aschematically depicts a device 60 according to an embodiment of theinvention that, similar to the above device 40, includes a conduit 42for receiving a flowing stream of an anti-solvent through a first inputport 44 as well as a second input port 50 for introducing a stream of apolymer solution into the mixed flowing stream of the anti-solvent. Indevice 60, a plurality of static mixer units 61 a, 61 b, 61 c aredisposed within the conduit 42 to cause mixing of the flowinganti-solvent. The static mixer units are disposed in series, preferablyin staggered orientation relative to one another. While FIG. 6Aillustrates the input port 50 as being positioned roughly in the centerof one of the static mixer units 61 b, in other embodiments the inputport 50 can be positioned differently. For example, in some embodiments,as illustrated in FIG. 6B, a device 62 can include a second input port50 that is positioned between adjacent static mixer units 61 a, 61 b soas to deliver the polymer solution to into a gap between those units.

FIG. 6C schematically illustrates a device 62′ according to anotherembodiment of the invention that includes a conduit 42 in which staticmixer units 61 a, 61 b are disposed. The device 62′ includes two inputports 50 a, 50 b, which are positioned on opposite sides of the conduit42, for the introduction of a polymer solution into the conduit. Thestatic mixer units are separated from one another so as to provide a gapin the vicinity of the input ports 50 a,50 b such that a polymersolution can be introduced through the gap into the conduit.

FIG. 7 schematically depicts a system 70 according to an embodiment ofthe invention for generating polymeric nanoparticles in which any of theabove devices 40, 52, 54, 56, 58, 58′, 58″, 60, 62 in any of theirvarious implementations can be incorporated. The system 70 includes areservoir 72 for storing a polymer solution and another reservoir 74 forstoring an anti-solvent, such as a mixture of deionized water and acolloid stabilizer. A device 78, e.g., a gear pump, is fluidly connectedat its input to the reservoir 74 and is fluidly coupled at its output tofirst input port 44 of the device 40 to cause a flow of the anti-solventfrom the reservoir into the conduit 42. A static mixer 48 is disposedwithin the conduit 42. As discussed above, the flow of the anti-solventstream through the static mixer generates a mixed flowing stream of theanti-solvent.

Another device 76, e.g., another gear pump, is fluidly connected at itsinput to the reservoir 72 and at is output to the second input port 50of the device 40 to cause a flow of the polymer solution from thereservoir 72 via the second port into the mixed flowing stream of theanti-solvent to cause precipitation of the polymer into a plurality ofpolymeric nanoparticles. As discussed above, although in manyembodiments the flow rate of the anti-solvent is substantially greaterthan that of the polymer solution, e.g. by a factor of 10, a variety ofanti-solvent and polymer solution flow rates can be employed.

In this embodiment, both of the devices 76 and 78 are variable pumpsthat can adjust the flow rate of the anti-solvent and the polymersolution, respectively, for introduction into the device 40. By way ofexample, in this implementation, the device 78 can adjust the flow rateof the anti-solvent in a range of about 20 ml/min to about 2000 ml/min,and the device 76 can adjust the flow rate of the polymer solution in arange of about 4 ml/min to about 200 ml/min, or, in some embodiments, ina range of about 5 ml/min to about 100 ml/min. As noted above, in someembodiments, the devices 76 and 78 are gear pumps. An example ofsuitable gear pump is a pump marketed by Cole-Parmer Instrument Companyof Illinois, U.S.A. under the trade designation Ismatec.

The output port 46 of the device 40 is in fluid communication with acollection vessel 84. The formed nanoparticles are entrained in a fluidstream comprising a mixture of the anti-solvent and the process solvent(in many cases the anti-solvent is the major component of the fluidstream) that carries the nanoparticles via the output port 46 into thecollection vessel 84, which may contain a liquid, e.g., deionized water.In some embodiments, the collection vessel is not pre-filled with aliquid. A suspension containing the nanoparticles can be collected fromthe collection vessel to be concentrated and in some embodimentslyophilized, e.g., in a manner discussed above.

As discussed above, it has been discovered that the use of a staticmixer to cause mixing of the anti-solvent stream allows generatingnanoparticles with a low polydispersity index over a wide range of flowrates, e.g., a polydispersity index equal to or less than about 0.25(e.g., in a range of about 0.05 to about 0.1). In addition, the flowrate can be adjusted to obtain a desired average particle size. In thisembodiment, the variable pump 78 allows changing the flow rate of theanti-solvent to “dial” the average particle size of the nanoparticlesgenerated via nanoprecipitation.

In some embodiments, one or more injectors can be employed to introducethe polymer solution into the mixed flowing stream of the anti-solvent.By way of example, FIG. 8A schematically shows a device 81 according tosuch an embodiment of the invention for generating nanoparticles, whichincludes a conduit 83 in which two static mixer units 85 a and 85 b aredisposed. Similar to the previous embodiments, the conduit 83 includesan input port 87 through which a fluid, e.g., anti-solvent, can beintroduced into the conduit and an output port 89 through which thefluid exits the conduit. In this embodiment, an injector 91 is coupledto the conduit at an intermediate location between the static mixerunits. The injector 91 includes an inlet port 93 for receiving a fluid,e.g., a polymer solution, and an output nozzle 95 that is positionedwithin the conduit and is configured to inject the fluid into the mixedflowing stream of the anti-solvent. Although the illustrated nozzlefaces downward (it is aimed substantially perpendicular to the axialdirection of the anti-solvent flow), the nozzle can also be aimed atother angles with respect the direction of the anti-solvent flow. Forexample, the nozzle can include a 90 degree bend such that it is aimedparallel to the direction of the anti-solvent flow towards the mixer'sdistal end.

FIG. 8B schematically depicts another device 97 according to theteachings of the invention that employs an injection system forinjecting a polymer solution into a mixed flowing stream of theanti-solvent. The device 97 includes a conduit 99 in which a two staticmixer 101 a and 101 b are disposed. An injection system 103 extendsacross the conduit within a gap between the two static mixer units. Theinjection system includes a plurality of injection nozzles 105 that areconfigured to inject a polymer solution in a downstream direction andwith sufficient velocity such that the polymer solution would beintroduced into a mixed flowing stream of an anti-solvent through themixer from mixer's proximal end to its distal end.

EXAMPLES

The following examples are provided for further elucidation of variousaspects of the invention. The examples are intended only forillustrative purposes and do not necessarily represent optimal ways ofpracticing the invention and/or optimal results that can be obtained.

A prototype system based on the system shown in FIG. 7 above wasassembled to generate polymeric nanoparticles in accordance with theabove teachings, as discussed in the following examples. In some of thefollowing examples, a helical static mixer similar to that shown in FIG.9 marketed by Cole-Parmer Instrument Company of Illinois, U.S.A. wasemployed. The helical static mixer includes alternating left andright-hand twists that cause a fluid flowing through the mixer to movefrom the wall of a conduit in which the mixer is disposed to the centerof the mixer and from the center to the wall in an alternating fashion.In some other examples, a static mixer known as a “structured-packingmixer” marketed by Sulzer Chemtech USA, Inc. of Oklahoma, U.S.A. underthe trade designation Sulzer SMX, shown in FIGS. 10A and 10B, wasemployed. This mixer includes a lattice of mixing elements that isoriented at 45 degrees relative to the direction of the flow.

At least two prototype devices were constructed:

I. A 5 mm internal diameter (ID) helical mixer device was constructed byinserting a 5 mm OD polyacetal helical mixer (Cole-Parmer) into a 5 mmID polypropylene tube fitted with a barbed polypropylene “Y” fitting onone end. (as shown in FIG. 11A). The mixer was extended through one ofthe arms of the “Y” fitting. The aqueous phase (i.e., anti-solvent) wasdirected via ¼ inch tubing through the mixer-containing arm. The organicphase (i.e., polymer solution) was directed via ⅛ inch tubing throughthe other (empty) arm. A ¼ inch to ⅛ inch reducer was connected to thebottom port to provide a slight back pressure.

II. An 8 mm ID modified SMX mixer device was constructed by modifying astandard ¼ inch Sulzer (Sulzer Chemtech USA, Tulsa, Okla.) SMX mixerwith an ⅛ inch ID side entry port midway along the static mixer length.The mixer was fabricated from 316L stainless steel mixer (FIG. 11B). Itwas 3 inches long with an 8 mm bored ID and contained 8 SMX typeelements. The mixer was configured such that the aqueous phase flowedthrough the mixer main body while the organic phase entered the sideport.

Example 1

The following process was used to characterize the effect of organic andaqueous flowrates on average particle size and polydispersity using theprototype helical mixing device depicted in FIG. 11A.

2 grams of 5050 PLGA (5050DLG 1AP, Mw=6.4 kD, Mn=2.9 kD, LakeshoreBiomaterials, Birmingham, Ala.) were dissolved in 158 grams of acetoneand sonicated for 30 seconds at room temperature. Separately, 10 gramsof poly vinyl alcohol (PVA) (80% hydrolyzed, Mw 9-10 kD, Aldrich, StLouis, Mo.) was dissolved in 2 kilograms of water at room temperature.Each solution was translucent and visually free of undissolved material.

The organic and aqueous phases were transferred to glass reservoirs, thebottom outlet of each was connected to pre-calibrated magneticallydriven gear pumps (Ismatec, Cole-Parmer) via flexible tubing to therespective ports of the helical mixer device. A particle size sample wascollected by first initiating the aqueous flow, then the organic flow topredetermined flow rates. After a few moments of flushing, a smallsuspension sample was collected at the tubing outlet and then pumps wereturned off in reverse order. The pump settings were then readjusted anda subsequent particle size sample taken. At the conclusion of theexperiment, each sample particle size was measured via Malvern ZetasizerModel Nano S (Malvern Instruments, Southborough, Mass.). In most caseseach particle size measurement was conducted in duplicate and theresults averaged.

Total flow rates in the range of about 25 to 500 ml/min with the organicphase relative to the aqueous phase flow rate (O:W) ratios of 1:5 and1:10 were employed and the results shown below:

TABLE 1 Qo Qw Qt Z-Avg D(v)50 D(v)90 D(v)10 O:W Ratio (ml/min) (ml/min)(ml/min) (nm) Pdl (nm) (nm) (nm) 1:5  73.0 365.0 438.0 84 0.089 68 10947 60.0 301.0 361.0 94 0.052 82 122 59 47.0 237.0 284.0 102 0.073 90 13863 34.3 172.0 206.3 111 0.093 97 156 67 21.5 107.4 128.9 123 0.059 112175 77 12.9 64.4 77.3 140 0.072 131 211 86 4.3 21.5 25.8 180 0.065 183268 126 1:10 46.4 463.5 509.9 87 0.082 71 114 48 38.6 386.5 425.1 920.071 78 121 54 30.1 300.0 330.1 98 0.091 81 134 55 21.5 214.0 235.5 1100.076 92 157 62 12.9 128.0 140.9 121 0.066 110 173 75 4.3 42.9 47.2 1520.061 147 232 97

In the above table and the tables that follow, Q_(o) and Q_(w) refer toflow rates of the organic and the aqueous phases, respectively. Q_(t)refers to the total flow rate. Dv50 is defined as the particle sizebelow which the sizes of 50% of the particles lie, Dv90 is defined asthe particle size below which the sizes of 90% of the particles lie, andDv10 is defined as the particle size below which the sizes of 10% of theparticles lie.

The polydispersity index remains less than about 0.1 over the entiretested flow rate range.

FIG. 12 presents data corresponding to Z_(ave) as a function of thetotal flow rate, indicating that the average particle size decreases asthe flow rate increases from about 25 ml/min to about 500 ml/min. Atflow rates less than about 200-300 ml/min, average particle sizedecreases at a much faster rate than flow rates greater than 200-300ml/min. In other words, two flow rate regimes can be discerned from thedata: one in which the average particle size is strongly flow dependentand another in which the average particle size can be considered assubstantially independent of the flow rate. FIG. 12 also shows thataverage particle sizes appear to be substantially independent of the O:Wratio within the range of 1:5 and 1:10.

These data show that the average particle size can be tuned (“dialed”)by changing the flow rates (principally the anti-solvent rate) throughthe mixer while ensuring that the polydispersity index remains low. Inother words, for a given desired average particle size, the flow ratescan be selected to achieve the target particle average size.

Example 2

The process described above in Example 1 was again conducted but thepolymer concentration in the polymer solution was increased to 2%. Datawas collected at both 1:5 and 1:10 O:W ratios.

The results are shown in the table below:

TABLE 2 O:W Qo Qw Qt Z-Avg D(v)50 D(v)90 D(v)10 ratio (ml/min) (ml/min)(ml/min) (nm) Pdl (nm) (nm) (nm) 1:5  73.0 365.0 438.0 110 0.058 97 15168 60.0 301.0 361.0 121 0.057 109 172 75 47.0 237.0 284.0 129 0.012 121177 87 34.3 172.0 206.3 137 0.059 129 200 88 21.5 107.4 128.9 157 0.042155 233 105 12.9 64.4 77.3 176 0.05 178 264 120 4.3 21.5 25.8 207 0.061214 307 150 1:10 46.4 463.5 509.9 119 0.058 108 166 75 38.6 386.5 425.1126 0.055 115 182 79 30.1 300.0 330.1 132 0.012 124 183 88 21.5 214.0235.5 140 0.059 133 203 93 12.9 128.0 140.9 155 0.053 152 232 101 4.342.9 47.2 191 0.026 194 283 134

As in Example 1, PdI was maintained at less than 0.1 for all flowconditions.

The Z_(ave) versus flow rate is plotted in FIG. 12 together with thedata in Example 1. The data shows that increasing the polymerconcentration from 1% to 2% results in an increase in the averageparticle size in a controlled fashion and with a similar dependency onthe flow rate as that observed for the 1% polymer concentration.

Example 3

The process discussed above in Example 1 was again conducted byutilizing the modified SMX mixer (shown in FIG. 11B) to test the effectof flow rate on average particle size. Using freshly prepared 1 wt %PLGA and 0.5 wt % PVA solutions and maintaining the W:O ratio at 10:1, aseries of experiments detailed below were conducted.

-   -   a) In the first experiment, the SMX mixer was utilized with a        set of pumps with a total flow rate range of 100 to 500 ml/min.    -   b) In the second experiment, the same mixer was used but with        another set of pumps with a larger flow rate in a range of 500        to 2000 ml/min.    -   c) In the third experiment, the helical mixer was used with the        pumps used in a) but at a single flow rate combination.    -   d) In the final experiment, the SMX mixer was used with pumps        used in b) but at 3 flow rate combinations.

The results are shown in the Table 3 below:

TABLE 3 Qo Qw Qt Z-Ave D10(v) D50(v) D90(v) Experiment (ml/min) (ml/min)(ml/min) (nm) Pdl (nm) (nm) (nm) a 10 100 110 232 0.115 151 250 425 15150 165 215 0.082 147 225 349 20 200 220 191 0.036 130 194 287 25 250275 185 0.068 123 188 290 30 300 330 191 0.100 111 197 338 35 350 385166 0.053 110 166 249 45 450 495 149 0.074 94 143 227 b 45 450 495 1680.057 112 168 252 60 660 660 152 0.062 97 148 231 75 750 825 140 0.06486 131 212 90 900 990 132 0.069 81 121 196 105 1050 1155 127 0.076 75114 190 120 1200 1320 123 0.053 77 112 174 135 1350 1485 115 0.077 67100 165 150 1500 1650 121 0.074 69 105 179 180 1800 1980 113 0.084 65 97164 C 45 450 495 121 0.151 59 97 199 45 450 495 128 0.025 84 119 178 D105 1050 1155 128 0.049 84 120 181 150 1500 1650 111 0.078 68 98 156 1801800 1980 107 0.062 63 91 147

The data shows that in most cases the PdI was maintained at less than0.1. A plot of Z_(ave) versus anti-solvent flow rate is shown in FIG.13. From this Figure, it can be seen that the data from the threeexperiments using the SMX mixer are comparable and may be represented bya single curve. The data obtained by utilizing the helical mixerindicates that smaller sized particles were generated compared to theparticles generated by utilizing the SMX mixer for the same flow rate.This can be due to a greater mixing intensity achieved in the helicalmixer device, which has a smaller diameter.

Example 4

Utilizing the helical mixer shown in FIG. 11A, the following process wasemployed to fabricate a 3-gram batch of docetaxel PEGylatednanoparticles.

2.52 grams of docetaxel custom conjugated PLGA (Mw=6.6 kD, Mn=3.0 kD,drug loading: 8%, AMRI Albany, N.Y.), and 0.480 grams of 5050DL-PLGAmPEG 2 kD (Mw: 10.6 kD, mPEG Mw: 2.0 kD, Lakeshore Biomaterials,Birmingham, Ala.) were dissolved in 237 grams of acetone at roomtemperature. Separately, a total of 15 grams of poly vinyl alcohol (PVA)(80% hydrolyzed, Mw 9-10 kD, Aldrich, St. Louis, Mo.) was dissolved in 3kilograms of water at room temperature. Each solution was translucentand visually free of undissolved material.

Referring to FIG. 7, the flow of the aqueous phase (anti-solvent) wasinitiated at a flow rate of 220 ml/min via the aqueous inlet through theconduit of the device in which the static mixer was disposed. Once theflow of the aqueous phase was established, the flow of the polymersolution was initiated at 22 ml/min from an organic solution vessel (notshown) through the organic inlet into the flowing stream of the aqueousphase. Once the organic solution vessel had emptied, the organic pumpwas turned off and the aqueous pump remained on for several moments toflush out the mixer. The recovered suspension collected in a collectionvessel was milky white and no large particles were distinguishable bythe naked eye.

The nanoparticle suspension was diafiltered and concentrated in thefollowing two-step process. The suspension was initially diafiltered toremove dissolved PVA and acetone by recirculation through a 300 kD MWCOfilter cassette (Pall Omega Centramate Medium Screen cassette, 0.093m²), at a recirculation flow rate of 200 ml/min and a transmembranepressure (TMP) of 1.5 bar using 44 L of deionized water. Uponcompletion, the nanoparticles suspension was drained from the cassetteand the cassette was rinsed twice by recirculating fresh deionized waterthrough the cassette and respective tubing (2×250 ml). The rinse volumeswere collected and combined with the initially recovered nanoparticlessuspension.

The washed nanoparticles suspension was subsequently concentrated usingthree smaller 300 kD MWCO filters (Millipore Pellicon XL, 50 cm² withBiomax membrane) plumbed in parallel at a recirculation rate of about 25ml/min and a TMP of 1 bar. Drug concentration was continuously monitoredby HPLC assay as the suspension volume was reduced. A portion of thesuspension was collected when the drug content level reached 1.7 mg/mland the remainder was collected at a drug content level of 3.2 mg/ml.Each suspension was stored at 4° C. The yield based on docetaxelrecovery was about 85.5%.

FIG. 14 shows the particle size distribution obtained by the Zetasizer.The average particle size (Z_(ave)) was measured to be about 97.5 nm,Dv90 (defined as the particle size below which the sizes of 90% of theparticles lies) was determined to be about 130.3 nm, and Dv50 (definedas the particle size below which the sizes of 50% of the particle lie)was determined to be about 85.2 nm. Further, the polydispersity index(PdI) was measured to be about 0.066.

Example 5

Utilizing the modified SMX mixer shown in FIG. 11B, the followingprocess was employed to fabricate a 10-gram batch of docetaxel PEGylatednanoparticles. 6.044 grams of docetaxel custom conjugated PLGA (Mw=9.8kD, Mn=5.7 kD, drug loading: 7.6%, AMRI Albany, N.Y.), and 4.003 gramsof 5050DL-PLGA mPEG 2 kD (Mw: 13.0 kD, mPEG Mw: 2.0 kD, LakeshoreBiomaterials, Birmingham, Ala.) were dissolved in 791 grams of acetoneat room temperature. Separately, 11 L of 0.5% solution of PVA wasprepared by combining 1.1 L of previously prepared stock solution of 5%PVA with 9.9 L of RODI water. (The stock solution was prepared bydissolving 110 gm of PVA (80% hydrolyzed, Mw: 9-10 kD, Sigma-Aldrich,St. Louis, Mo.) into 2200 ml of RODI water and heating the solution at80° C. for 3 hr. The solution was cooled to room temperature, filteredand stored at 4° C.

Referring to FIG. 7, the flow of the aqueous phase (anti-solvent) wasinitiated at a flow rate of 608 ml/min via the aqueous inlet through theconduit of the device in which the static mixer was disposed. Once theflow of the aqueous phase was established, the flow of the polymersolution was initiated at 60.8 ml/min from an organic solution vessel(not shown) through the organic inlet into the flowing stream of theaqueous phase. Once the organic solution vessel was empty, the organicpump was turned off and the aqueous pump remained on for several momentsto flush out the mixer. The recovered suspension, approximately 11liters, was collected in 20 liter polypropylene carboy.

The nanoparticle suspension was diafiltered and concentrated using atangential flow filter (TFF) (GE Healthcare hollow fiber cartridge,polysulfone membrane, 500 kD NMWC, 0.14 m²) in a three-step process.Using a recirculation rate of 1160 ml/min and a transmembrane pressure(TMP) of less than 20 psi, the suspension was initially concentrated toapproximately 1 liter, diafiltered with 12 liters of RODI water, andconcentrated a final time to 206 ml. The suspension was recovered andmeasured by HPLC for docetaxel content and stored at 4° C. The yieldbased on docetaxel recovery was about 96%.

FIG. 15 shows three graphs corresponding to three measurements, obtainedby Malvern Zetasizer Model Nano S (Malvern Instruments, Southborough,Mass.), of the particle size distribution of the nanoparticles. Theaverage particle size (Z_(ave)) was measured to be about 80.35 nm, Dv90(defined as the particle size below which the sizes of 90% of theparticles lies) was determined to be about 103 nm, and Dv50 (defined asthe particle size below which the sizes of 50% of the particle lie) wasdetermined to be about 69.2 nm. Further, the polydispersity index (PdI)was measured to be 0.052.

All publications referred to herein, including patents, published patentapplications, articles, among others, are hereby incorporated byreference in their entirety.

Those having ordinary skill in the art will appreciate that variouschanges can be made to the above embodiments without departing from thescope of the invention.

What is claimed is:
 1. A population of polymeric nanoparticles,comprising: a plurality of polymeric nanoparticles having an amphiphilicco-polymer as at least one constituent, wherein said amphiphilicco-polymer includes polylactic acid (PLA) as one of its polymericcomponents, wherein said polymeric nanoparticles exhibit an averageparticle size equal to or less than about 100 nm and a polydispersityindex in a range of about 0.05 and about 0.1.
 2. The population of thepolymeric nanoparticles of claim 1, wherein said polymeric nanoparticlesexhibit an average particle size equal to or less than about 95 nm. 3.The population of the polymeric nanoparticles of claim 1, wherein saidpolymeric nanoparticles exhibit an average particle size equal to orless than about 90 nm.
 4. The population of the polymeric nanoparticlesof claim 1, wherein said polymeric nanoparticles exhibit an averageparticle size equal to or less than about 85 nm.
 5. The population ofthe polymeric nanoparticles of claim 1, wherein said polymericnanoparticles exhibit an average particle size equal to or less thanabout 80 nm.
 6. The population of the polymeric nanoparticles of claim1, wherein said polymeric nanoparticles exhibit an average particle sizeequal to or less than about 75 nm.
 7. The population of the polymericnanoparticles of claim 1, wherein said polymeric nanoparticles exhibitan average particle size equal to or less than about 70 nm.
 8. Thepopulation of the polymeric nanoparticles of claim 1, wherein saidpolymeric nanoparticles exhibit an average particle size equal to orless than about 65 nm.
 9. The population of the polymeric nanoparticlesof claim 1, wherein said polymeric nanoparticles exhibit an averageparticle size equal to or less than about 60 nm.
 10. The population ofthe polymeric nanoparticles of claim 1, wherein said polymericnanoparticles exhibit an average particle size equal to or less thanabout 55 nm.
 11. The population of the polymeric nanoparticles of claim1, wherein said polymeric nanoparticles exhibit an average particle sizeequal to or less than about 50 nm.
 12. The population of the polymericnanoparticles of claim 1, wherein said polymeric nanoparticles compriseany of a therapeutic agent or an imaging agent.
 13. The population ofthe polymeric nanoparticles of claim 12, wherein said therapeutic agentis any of a taxane, an epothilone, a boronic acid proteasome inhibitor,and an anti-biotic.
 14. The population of the polymeric nanoparticles ofclaim 12, wherein said therapeutic agent is an anti-neoplastic agent.15. The population of the polymeric nanoparticles of claim 14, whereinsaid anti-neoplastic agent is a taxane.
 16. The population of thepolymeric nanoparticles of claim 15, wherein said taxane is any ofpaclitaxel, docetaxel, larotaxel and cabazitaxel.
 17. The population ofthe polymeric nanoparticles of claim 1, wherein said population includesat least about 10 grams of said nanoparticles.
 18. The population of thepolymeric nanoparticles of claim 1, wherein said population includes atleast about 50 grams of said nanoparticles.
 19. The population of thepolymeric nanoparticles of claim 1, wherein said population includes atleast about 100 grams of said nanoparticles.
 20. The population of thepolymeric nanoparticles of claim 1, wherein said population includes atleast about 500 grams of said nanoparticles.
 21. The population of thepolymeric nanoparticles of claim 1, wherein said population comprises alyoprotectant.
 22. A population of polymeric nanoparticles, comprising:a plurality of polymeric nanoparticles having an amphiphilic co-polymeras at least one constituent, wherein said amphiphilic co-polymerincludes polyglycolic acid (PGA) as one of its polymeric components,wherein said polymeric nanoparticles exhibit an average particle sizeequal to or less than about 100 nm and a polydispersity index in a rangeof about 0.05 and about 0.1.
 23. The population of the polymericnanoparticles of claim 22, wherein said polymeric nanoparticles exhibitan average particle size equal to or less than about 95 nm.
 24. Thepopulation of the polymeric nanoparticles of claim 22, wherein saidpolymeric nanoparticles exhibit an average particle size equal to orless than about 90 nm.
 25. The population of the polymeric nanoparticlesof claim 22, wherein said polymeric nanoparticles exhibit an averageparticle size equal to or less than about 85 nm.
 26. The population ofthe polymeric nanoparticles of claim 22, wherein said polymericnanoparticles exhibit an average particle size equal to or less thanabout 80 nm.
 27. The population of the polymeric nanoparticles of claim22, wherein said polymeric nanoparticles exhibit an average particlesize equal to or less than about 75 nm.
 28. The population of thepolymeric nanoparticles of claim 22, wherein said polymericnanoparticles exhibit an average particle size equal to or less thanabout 70 nm.
 29. The population of the polymeric nanoparticles of claim22, wherein said polymeric nanoparticles exhibit an average particlesize equal to or less than about 65 nm.
 30. The population of thepolymeric nanoparticles of claim 22, wherein said polymericnanoparticles exhibit an average particle size equal to or less thanabout 60 nm.
 31. The population of the polymeric nanoparticles of claim22, wherein said polymeric nanoparticles exhibit an average particlesize equal to or less than about 55 nm.
 32. The population of thepolymeric nanoparticles of claim 22, wherein said polymericnanoparticles exhibit an average particle size equal to or less thanabout 50 nm.
 33. The population of the polymeric nanoparticles of claim22, wherein said polymeric nanoparticles comprise any of a therapeuticagent or an imaging agent.
 34. The population of the polymericnanoparticles of claim 22, wherein said therapeutic agent is any of ataxane, an epothilone, a boronic acid proteasome inhibitor, and ananti-biotic.
 35. The population of the polymeric nanoparticles of claim32, wherein said therapeutic agent is an anti-neoplastic agent.
 36. Thepopulation of the polymeric nanoparticles of claim 33, wherein saidanti-neoplastic agent is a taxane.
 37. The population of the polymericnanoparticles of claim 34, wherein said taxane is any of paclitaxel,docetaxel, larotaxel and cabazitaxel.
 38. The population of thepolymeric nanoparticles of claim 22, wherein said population includes atleast about 10 grams of said nanoparticles.
 39. The population of thepolymeric nanoparticles of claim 22, wherein said population includes atleast about 50 grams of said nanoparticles.
 40. The population of thepolymeric nanoparticles of claim 22, wherein said population includes atleast about 100 grams of said nanoparticles.
 41. The population of thepolymeric nanoparticles of claim 22, wherein said population includes atleast about 500 grams of said nanoparticles.
 42. The population of thepolymeric nanoparticles of claim 22, wherein said population comprises alyoprotectant.